{"id":733,"date":"2017-11-09T14:31:09","date_gmt":"2017-11-09T14:31:09","guid":{"rendered":"https:\/\/lab.research.sickkids.ca\/fcf\/?page_id=733"},"modified":"2019-08-30T15:27:18","modified_gmt":"2019-08-30T15:27:18","slug":"faq","status":"publish","type":"page","link":"https:\/\/lab.research.sickkids.ca\/fcf\/faq\/","title":{"rendered":"FAQ"},"content":{"rendered":"<div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-0 fusion_builder_column_5_6 5_6 fusion-five-sixth fusion-column-first\" style=\"--awb-bg-size:cover;width:83.333333333333%;width:calc(83.333333333333% - ( ( 4% ) * 0.83333333333333 ) );margin-right: 4%;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><style type=\"text\/css\">.fusion-faqs-wrapper #accordian-1 .fusion-panel { border-color:#e0dede; }.fusion-faqs-wrapper #accordian-1 .fusion-panel:hover{ border-color: #e0dede; }.fusion-accordian #accordian-1 .panel-title a .fa-fusion-box:before{ font-size: 13px;width: 13px;}.fusion-accordian #accordian-1 .panel-title a .fa-fusion-box{ color: #ffffff;}.fusion-accordian  #accordian-1 .panel-title a{font-family:Arial, Helvetica, sans-serif;font-style:normal;font-weight:400;}.fusion-accordian  #accordian-1 .panel-title a:not(:hover){}.fusion-accordian  #accordian-1 .toggle-content{font-family:Arial, Helvetica, sans-serif;font-style:normal;font-weight:400;}.fusion-accordian #accordian-1 .fa-fusion-box { background-color: #333333 !important;border-color: #333333 !important;}.fusion-accordian #accordian-1 .panel-title a:hover,.fusion-accordian #accordian-1 .panel-title a.hover { color: #d4ba00;}.fusion-faq-shortcode .fusion-accordian #accordian-1 .fusion-toggle-boxed-mode:hover .panel-title a { color: #d4ba00;}.fusion-accordian #accordian-1 .panel-title .active .fa-fusion-box,.fusion-accordian #accordian-1 .panel-title a:hover .fa-fusion-box,.fusion-accordian #accordian-1 .panel-title a.hover .fa-fusion-box { background-color: #d4ba00!important;border-color: #d4ba00!important;}<\/style><div class=\"fusion-faq-shortcode\" style=\"\"><ul role=\"menu\" class=\"fusion-filters clearfix\"><li role=\"presentation\" class=\"fusion-filter fusion-active\"><a role=\"menuitem\" data-filter=\".cell-sorting\" href=\"#\">Cell sorting<\/a><\/li><li role=\"presentation\" class=\"fusion-filter fusion-hidden\"><a role=\"menuitem\" data-filter=\".troubleshooting\" href=\"#\">Troubleshooting<\/a><\/li><\/ul><div class=\"fusion-faqs-wrapper\"><div class=\"accordian fusion-accordian\"><div class=\"panel-group \" id=\"accordian-1\"><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-766 troubleshooting \"><span class=\"entry-title rich-snippet-hidden\">Droplet containment vacuum not functioning<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T20:55:31+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-766\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-766\" href=\"#collapse-1-766\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">Droplet containment vacuum not functioning<\/div><\/a><\/h4><\/div><div id=\"collapse-1-766\" aria-labelledby=\"faq_1-766\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-1 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-1 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-1\">\n<table id=\"tablepress-29\" class=\"tablepress tablepress-id-29 tablepress-responsive tbody-has-connected-cells\">\n<thead>\n<tr class=\"row-1\">\n\t<th class=\"column-1\"><strong>Observation<\/strong><\/th><th class=\"column-2\"><strong>Possible causes<\/strong><\/th><th class=\"column-3\"><strong>Recommended solutions<\/strong><\/th>\n<\/tr>\n<\/thead>\n<tbody class=\"row-striping row-hover\">\n<tr class=\"row-2\">\n\t<td rowspan=\"5\" class=\"column-1\">Droplet containment vacuum not functioning<\/td><td class=\"column-2\">Worn O-ring in retainer<\/td><td class=\"column-3\">Replace the O-ring. Contact FMCF staff.<\/td>\n<\/tr>\n<tr class=\"row-3\">\n\t<td class=\"column-2\">Outer sleeve is not seated in the retainer<\/td><td class=\"column-3\">1. Loosen the retainer<br \/>\n2. Push the outer sleeve up into the retainer until seated.<br \/>\n3. Tighten the retainer<br \/>\n<\/td>\n<\/tr>\n<tr class=\"row-4\">\n\t<td class=\"column-2\">Outer sleeve is not on the sample injection tube<\/td><td class=\"column-3\">Replace the outer sleeve.<br \/>\n1. Loosen the retainer.<br \/>\n2. Slide the outer sleeve over the sample injection tube until it is seated.<br \/>\n3. Tighten the retainer.<\/td>\n<\/tr>\n<tr class=\"row-5\">\n\t<td class=\"column-2\">Waste line is pinched, preventing proper aspiration<\/td><td class=\"column-3\">Check the waste line.<\/td>\n<\/tr>\n<tr class=\"row-6\">\n\t<td class=\"column-2\">Waste tank is full<\/td><td class=\"column-3\">Empty the waste tank.<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<!-- #tablepress-29 from cache -->\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-735 cell-sorting \"><span class=\"entry-title rich-snippet-hidden\">How many cells do I need to bring to the sorter?<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2018-01-23T19:20:54+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-735\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-735\" href=\"#collapse-1-735\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">How many cells do I need to bring to the sorter?<\/div><\/a><\/h4><\/div><div id=\"collapse-1-735\" aria-labelledby=\"faq_1-735\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-2 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-2 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-2\"><p>This is the most commonly asked question. To answer it we need to know the following information:<\/p>\n<ol>\n<li>What is the approximate percentage of the population(s) you wish to sort?<\/li>\n<li>How many total cells do you want back?<\/li>\n<li>Do you want stringent purification or enrichment?<\/li>\n<li>How fragile or large are your cells?<\/li>\n<\/ol>\n<p>All of these parameters influence the yield and purity of cell sorting. Here is an example: Let&#8217;s say the subset you want sorted is 20% of the total and you need 2 million cells for your experiment. In theory you would need to run 10 million cells through the sorter (10 million x 20% = 2 million). However, the actual yield is usually 75-95% of this theoretical yield, due to abort rates (caused by sort conflicts) and also the quality of your sample.<\/p>\n<p>Therefore, we recommend that you bring 25-50% more cells to the sorter than you would need if the actual yield were 100% (based on the abundance of your target cells).<\/p>\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-777 troubleshooting \"><span class=\"entry-title rich-snippet-hidden\">Sample tube not fitting on SIP<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2018-07-27T15:46:33+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-777\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-777\" href=\"#collapse-1-777\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">Sample tube not fitting on SIP<\/div><\/a><\/h4><\/div><div id=\"collapse-1-777\" aria-labelledby=\"faq_1-777\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-3 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-3 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-3\">\n<table id=\"tablepress-30\" class=\"tablepress tablepress-id-30 tablepress-responsive tbody-has-connected-cells\">\n<thead>\n<tr class=\"row-1\">\n\t<th class=\"column-1\"><strong>Observation<\/strong><\/th><th class=\"column-2\"><strong>Possible causes<\/strong><\/th><th class=\"column-3\"><strong>Recommended solutions<\/strong><\/th>\n<\/tr>\n<\/thead>\n<tbody class=\"row-striping row-hover\">\n<tr class=\"row-2\">\n\t<td rowspan=\"2\" class=\"column-1\">Sample tube not fitting on SIP<\/td><td class=\"column-2\">Sample tube other than BD Falcon tubes used<\/td><td class=\"column-3\">Use BD Falcon 12 x 75-mm sample tubes.<br \/>\nTubes must be polystyrene NOT polypropylene<\/td>\n<\/tr>\n<tr class=\"row-3\">\n\t<td class=\"column-2\">Worn Bal seal<\/td><td class=\"column-3\">Replace the Bal seal. Contact FMCF staff<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<!-- #tablepress-30 from cache -->\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-739 cell-sorting \"><span class=\"entry-title rich-snippet-hidden\">How long will it take to sort?<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T14:58:29+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-739\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-739\" href=\"#collapse-1-739\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">How long will it take to sort?<\/div><\/a><\/h4><\/div><div id=\"collapse-1-739\" aria-labelledby=\"faq_1-739\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-4 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-4 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-4\"><p>The answer to this question depends primarily on your goal (enrichment vs stringent purification), the nature of your cells (fragile cells or large cells need to be sorted at lower pressures and speeds with a larger nozzle), and the concentration of your sample (more dilute samples will take longer to sort).<\/p>\n<p>For example, on the MoFlo cell sorter, we use the 100\u00b5m nozzle at 30psi. For primary lymphocytes we run samples at maximum flow rates of 10,000-13,000\/second. For cell lines, we run samples 400- 4,000\/second depending on the size of the cells. With the Aria cell sorters, we can run primary lymphocytes at 6,000-8,000\/second with the 100 \u00b5m nozzle at 30psi, or at 9,000-10,000\/second with the 70 \u00b5m nozzle at 60psi.<\/p>\n<p>Again, for cell lines the flow rates will vary a lot depending on the size of cells. For example, if your cells are run at 10,000\/second, it would take approximately 30 minutes to run a total of 18 million cells through the sorter. Using the above scenario, your theoretical yield is 18 million x 20% = 3.6 million. If you require stringent purity or your sample is of poor quality (low viability, clumping, etc), your actual yield will be lower.<\/p>\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-741 cell-sorting \"><span class=\"entry-title rich-snippet-hidden\">Do I need to filter my cells?<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T14:57:55+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-741\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-741\" href=\"#collapse-1-741\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">Do I need to filter my cells?<\/div><\/a><\/h4><\/div><div id=\"collapse-1-741\" aria-labelledby=\"faq_1-741\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-5 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-5 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-5\"><p>It is REQUIRED that your cells be filtered through at 70um nylon mesh, preferably right before running on the sorter. We can provide the mesh if necessary.<\/p>\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-781 troubleshooting \"><span class=\"entry-title rich-snippet-hidden\">Rapid sample aspiration<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T20:55:46+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-781\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-781\" href=\"#collapse-1-781\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">Rapid sample aspiration<\/div><\/a><\/h4><\/div><div id=\"collapse-1-781\" aria-labelledby=\"faq_1-781\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-6 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-6 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-6\">\n<table id=\"tablepress-31\" class=\"tablepress tablepress-id-31 tablepress-responsive tbody-has-connected-cells\">\n<thead>\n<tr class=\"row-1\">\n\t<th class=\"column-1\"><strong>Observation<\/strong><\/th><th class=\"column-2\"><strong>Possible causes<\/strong><\/th><th class=\"column-3\"><strong>Recommended solutions<\/strong><\/th>\n<\/tr>\n<\/thead>\n<tbody class=\"row-striping row-hover\">\n<tr class=\"row-2\">\n\t<td rowspan=\"2\" class=\"column-1\">Rapid sample aspiration<\/td><td class=\"column-2\">Support arm is to the side<\/td><td class=\"column-3\">Place the support arm under the sample tube<\/td>\n<\/tr>\n<tr class=\"row-3\">\n\t<td class=\"column-2\">Droplet containment module is failing<\/td><td class=\"column-3\">Call your service representative. Contact FMCF staff<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<!-- #tablepress-31 from cache -->\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-744 cell-sorting \"><span class=\"entry-title rich-snippet-hidden\">What do I re-suspend my cells in for sorting?<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2023-06-20T18:39:54+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-744\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-744\" href=\"#collapse-1-744\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">What do I re-suspend my cells in for sorting?<\/div><\/a><\/h4><\/div><div id=\"collapse-1-744\" aria-labelledby=\"faq_1-744\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-7 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-7 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-7\"><p><strong>Proper sample preparation is absolutely essential for obtaining good purity and yields.<\/strong> After the final wash, cells should be re-suspended at concentrations of 20-30 million (for primary cells) or 5-10 million (for cell lines) per mL in FACS Tubes (see below). The media can be any buffered isotonic salt solution (such as PBS) but we recommend Hanks Balanced Salt Solution (HBSS) <strong>calcium and magnesium free<\/strong> containing 10 mM HEPES, pH 7.2).<\/p>\n<p>Most cells benefit from having some protein in the media, such as 1-2% FBS, calf serum or BSA. Higher serum concentrations can lead to cell aggregation and clogging. For cell line samples, we recommend also adding 1mM EDTA. We recommend using buffers that do not contain phenol red because phenol red causes an increase in background fluorescence, and reduces sensitivity. This effect is particularly bad for dim green signals.<\/p>\n<p><strong>For sticky cells:<\/strong><em>\u00a0<\/em> Increase the concentration of the EDTA to 5mM and use\u00a0FBS that has been dialyzed against Ca\/Mg++ free PBS or 0.5% BSA. Some cell types, however, can be sensitive to high concentrations of EDTA.<\/p>\n<p><strong>For adherent cells:<\/strong><em>\u00a0\u00a0<\/em>To detach adherent cells, trypsin (or other detachment buffer) is often quenched with media or buffer containing serum.\u00a0 This reintroduces cations that can promote aggregation. Use\u00a0FBS that has been dialyzed against Ca\/Mg++ free PBS or increase the EDTA concentration to 5mM or higher (Some cell types, however, can be sensitive to high concentrations of EDTA.). Accutase and Accumax are cell detachment solutions\u00a0that can be used to generate single cell suspensions.<\/p>\n<p><strong>NOTE:<\/strong> Accutase can alter some cell surface epitopes; the effect will need to be determined empirically for the epitopes being evaluated.<\/p>\n<p><strong>For samples with a high percentage of dead cells:<\/strong><em>\u00a0<\/em> DNA from dead cells can cause cell clumping. DNAse I in the presence of magnesium chloride will help reduce cellular aggregation.<\/p>\n<ol class=\"la\">\n<li>Treat cells for 15 to 30 minutes in a solution of 100 \u00b5g\/mL DNAse and 5 mM MgCl2 in HBSS at room temperature.<\/li>\n<li>Wash the cells once in HBSS with 5 mM MgCl<sub>2<\/sub>.<\/li>\n<li>Gently resuspend the cells in HBSS with 5 mM MgCl<sub>2<\/sub> and add 25-50 \u00b5g\/mL DNAse (as a maintenance dose) prior to and during the sort.<\/li>\n<\/ol>\n<p><strong>Note:<\/strong> DNAse I requires at least 1 mM magnesium to work effectively, although 5mM is optimal.\u00a0It is important to minimize the presence of dead cells during this procedure, since actin released from dead cells irreversibly inhibits DNAse I.<\/p>\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-757 cell-sorting \"><span class=\"entry-title rich-snippet-hidden\">What controls do I need to bring?<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2018-07-27T15:46:13+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-757\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-757\" href=\"#collapse-1-757\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">What controls do I need to bring?<\/div><\/a><\/h4><\/div><div id=\"collapse-1-757\" aria-labelledby=\"faq_1-757\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-8 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-8 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-8\"><p>You will need to bring an unstained control, single colour compensation controls and you MAY need to bring FMO (Fluorescence Minus One) gating controls. You may want to consider an FMO control if you are using more than 5 colours simultaneously and if your populations of interest cannot be discretely separated from background\/negatives.<\/p>\n<p>Please refer to the table below to see an example of a typical sort experiment with three colours.<\/p>\n\n<table id=\"tablepress-28\" class=\"tablepress tablepress-id-28 tablepress-responsive tbody-has-connected-cells\">\n<thead>\n<tr class=\"row-1\">\n\t<th class=\"column-1\"><Strong>Control type<\/strong><\/th><th class=\"column-2\"><Strong>Sample Name<\/strong><\/th><th class=\"column-3\"><Strong>Marker +<br \/>\nfluorochrome #1<\/strong><\/th><th class=\"column-4\"><Strong>Marker + <br \/>\nfluorochrome #2<\/strong><\/th><th class=\"column-5\"><Strong>Marker + <br \/>\nfluorochrome #3<\/strong><\/th>\n<\/tr>\n<\/thead>\n<tbody class=\"row-striping row-hover\">\n<tr class=\"row-2\">\n\t<td class=\"column-1\">Unstained Control<\/td><td class=\"column-2\">Unstained<\/td><td class=\"column-3\">-<\/td><td class=\"column-4\">-<\/td><td class=\"column-5\">-<\/td>\n<\/tr>\n<tr class=\"row-3\">\n\t<td rowspan=\"3\" class=\"column-1\">Single colour compensation controls<\/td><td class=\"column-2\">CD3 - FITC<\/td><td class=\"column-3\">CD3 - FITC<\/td><td class=\"column-4\">-<\/td><td class=\"column-5\">-<\/td>\n<\/tr>\n<tr class=\"row-4\">\n\t<td class=\"column-2\">CD4 - PE<\/td><td class=\"column-3\">-<\/td><td class=\"column-4\">CD4 - PE<\/td><td class=\"column-5\">-<\/td>\n<\/tr>\n<tr class=\"row-5\">\n\t<td class=\"column-2\">CD8 - APC<\/td><td class=\"column-3\">-<\/td><td class=\"column-4\"><\/td><td class=\"column-5\">CD8 - APC<\/td>\n<\/tr>\n<tr class=\"row-6\">\n\t<td class=\"column-1\">FMO Control<\/td><td class=\"column-2\">FMO FITC<\/td><td class=\"column-3\">-<\/td><td class=\"column-4\">CD4 - PE<\/td><td class=\"column-5\">CD8 - APC<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n<p><strong>Fluorophores excited by the Yellow\/Green laser<\/strong><br \/>\nIf you are using markers such as mCherry, RFP, Tomato Red, Texas-Red, etc, Please contact the Flow Facility to inquire about the specific sorter to book as only a limited number of sorters are equipped with an appropriate laser to detect these signals.<\/p>\n<p><strong>Viability Dyes<\/strong><br \/>\nWe recommend that you add a viability dye such as PI, 7AAD, or LIVE\/DEAD fixable dyes (Invitrogen) to your sample to enable exclusion of dead cells during sorting. If you choose to do so, please bring an appropriate single color compensation control. We suggest the following: Heat killed cells (65oC for 15 minutes and then mix with live cells) followed by staining with viability dye.<\/p>\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-747 troubleshooting \"><span class=\"entry-title rich-snippet-hidden\">What are FACS Tubes and where do I get them?<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T20:55:03+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-747\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-747\" href=\"#collapse-1-747\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">What are FACS Tubes and where do I get them?<\/div><\/a><\/h4><\/div><div id=\"collapse-1-747\" aria-labelledby=\"faq_1-747\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-9 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-9 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-9\"><p>BD Falcon makes FACS Tubes of various kinds. For all cell sorters, order 5 ml sterile polypropylene tubes (catalogue number BD2063). For non-sterile and analytical samples order catalogue number BD2002 (polypropylene) or BD2052 (polystyrene).<\/p>\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-753 cell-sorting \"><span class=\"entry-title rich-snippet-hidden\">What kind of tubes or plates should the cells be sorted into?<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T14:59:05+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-753\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-753\" href=\"#collapse-1-753\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">What kind of tubes or plates should the cells be sorted into?<\/div><\/a><\/h4><\/div><div id=\"collapse-1-753\" aria-labelledby=\"faq_1-753\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-10 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-10 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-10\"><p>For abundant populations we recommend 50 mL (MoFlo) or 15 mL (FACS Aria) Falcon Tubes, or 5 mL Falcon tubes. We can also sort into a range of microtiter plates, including from 96-well to 8-well, microscope slides or Petri dishes. Please talk to us about your requirements and we will advise you on the best choice for your experiment.<\/p>\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-785 troubleshooting \"><span class=\"entry-title rich-snippet-hidden\">No events in acquisition display and RUN button is green<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T20:55:54+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-785\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-785\" href=\"#collapse-1-785\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">No events in acquisition display and RUN button is green<\/div><\/a><\/h4><\/div><div id=\"collapse-1-785\" aria-labelledby=\"faq_1-785\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-11 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-11 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-11\">\n<table id=\"tablepress-32\" class=\"tablepress tablepress-id-32 tablepress-responsive tbody-has-connected-cells\">\n<thead>\n<tr class=\"row-1\">\n\t<th class=\"column-1\"><strong>Observation<\/strong><\/th><th class=\"column-2\"><strong>Possible causes<\/strong><\/th><th class=\"column-3\"><strong>Recommended solutions<\/strong><\/th>\n<\/tr>\n<\/thead>\n<tbody class=\"row-striping row-hover\">\n<tr class=\"row-2\">\n\t<td rowspan=\"17\" class=\"column-1\">No events in acquisition display and RUN button is green<\/td><td class=\"column-2\">Threshold is not set to the correct parameter (usually FSC)<\/td><td class=\"column-3\">Set the threshold to the correct parameter for your application<\/td>\n<\/tr>\n<tr class=\"row-3\">\n\t<td class=\"column-2\">Threshold level is too high<\/td><td class=\"column-3\">Lower the threshold level<\/td>\n<\/tr>\n<tr class=\"row-4\">\n\t<td class=\"column-2\">PMT voltage for threshold parameter is set too low<\/td><td class=\"column-3\">Set the PMT voltage higher for the threshold parameter<\/td>\n<\/tr>\n<tr class=\"row-5\">\n\t<td class=\"column-2\">Gating issue<\/td><td class=\"column-3\">Ensure gating strategy is appropriate<\/td>\n<\/tr>\n<tr class=\"row-6\">\n\t<td class=\"column-2\">Air in the sheath filter<\/td><td class=\"column-3\">Purge the filter<\/td>\n<\/tr>\n<tr class=\"row-7\">\n\t<td class=\"column-2\">Sheath and air line not connected to the sheath tank<\/td><td class=\"column-3\">Ensure sheath and air line are connected to the sheath tank<\/td>\n<\/tr>\n<tr class=\"row-8\">\n\t<td class=\"column-2\">O-ring is not in position in the sheath tank<\/td><td class=\"column-3\">Reposition O-ring in sheath tank<\/td>\n<\/tr>\n<tr class=\"row-9\">\n\t<td class=\"column-2\">Cracked tube\/ Bal seal (tube to SIP connection) problem<\/td><td class=\"column-3\">Replace tube<\/td>\n<\/tr>\n<tr class=\"row-10\">\n\t<td class=\"column-2\">No sample in the tube<\/td><td class=\"column-3\">Add sample to the tube or install a new sample tube<\/td>\n<\/tr>\n<tr class=\"row-11\">\n\t<td class=\"column-2\">Sample is not mixed properly<\/td><td class=\"column-3\">Mix the sample to suspend cells<\/td>\n<\/tr>\n<tr class=\"row-12\">\n\t<td class=\"column-2\">Waste tank is full<\/td><td class=\"column-3\">Empty the waste tank<\/td>\n<\/tr>\n<tr class=\"row-13\">\n\t<td class=\"column-2\">PMT voltages set too low or too high for display parameter<\/td><td class=\"column-3\">Reset the PMT voltages<\/td>\n<\/tr>\n<tr class=\"row-14\">\n\t<td class=\"column-2\">Too few events are displayed<\/td><td class=\"column-3\">Increase the number of events to display<\/td>\n<\/tr>\n<tr class=\"row-15\">\n\t<td class=\"column-2\">Sample injection tube is clogged<\/td><td class=\"column-3\">Remove the sample tube to allow backflushing and prime the instrument several times. Clean instrument. Contact FMCF staff if issue is not resolved<\/td>\n<\/tr>\n<tr class=\"row-16\">\n\t<td class=\"column-2\">Laser is not warmed up<\/td><td class=\"column-3\">Wait the recommended amount of time for the laser to warm up.<br \/>\n- 30 min for the 488-nm (blue)<br \/>\n- 30 min for the 355-nm (UV)<br \/>\n- 15 min for the 405-nm (violet)<br \/>\n- 20 min for the 633-nm (red)<\/td>\n<\/tr>\n<tr class=\"row-17\">\n\t<td class=\"column-2\">Laser delay is set incorrectly<\/td><td class=\"column-3\">Contact FMCF staff<\/td>\n<\/tr>\n<tr class=\"row-18\">\n\t<td class=\"column-2\">Laser is not functioning<\/td><td class=\"column-3\">Verify the malfunction by changing the threshold to an alternative laser while running the appropriate sample. If unsuccessful, contact FMCF staff<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<!-- #tablepress-32 from cache -->\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-755 cell-sorting \"><span class=\"entry-title rich-snippet-hidden\">What collection medium should the cells be sorted into?<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T14:59:16+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-755\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-755\" href=\"#collapse-1-755\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">What collection medium should the cells be sorted into?<\/div><\/a><\/h4><\/div><div id=\"collapse-1-755\" aria-labelledby=\"faq_1-755\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-12 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-12 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-12\"><p>We recommend 100% serum or HEPES buffered HBSS.<\/p>\n<p>The sorter uses PBS as its sheath fluid. Mixing large quantities of PBS with buffer containing calcium chloride may produce a precipitate if calcium phosphate on the cell membranes. This can adversely affect viability. For this reason, it is preferable to sort large numbers of cells into 100% serum, and not into regular culture medium. HEPES buffered HBSS without calcium is an alternative. If you must sort into medium, remember that the pH will rise with time, and it&#8217;s best to start with it on the acid side of neutral.<\/p>\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-793 troubleshooting \"><span class=\"entry-title rich-snippet-hidden\">No events in acquisition display and RUN button is orange<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T20:56:01+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-793\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-793\" href=\"#collapse-1-793\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">No events in acquisition display and RUN button is orange<\/div><\/a><\/h4><\/div><div id=\"collapse-1-793\" aria-labelledby=\"faq_1-793\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-13 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-13 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-13\">\n<table id=\"tablepress-33\" class=\"tablepress tablepress-id-33 tablepress-responsive tbody-has-connected-cells\">\n<thead>\n<tr class=\"row-1\">\n\t<th class=\"column-1\"><strong>Observation<\/strong><\/th><th class=\"column-2\"><strong>Possible causes<\/strong><\/th><th class=\"column-3\"><strong>Recommended solutions<\/strong><\/th>\n<\/tr>\n<\/thead>\n<tbody class=\"row-striping row-hover\">\n<tr class=\"row-2\">\n\t<td rowspan=\"8\" class=\"column-1\">No events in acquisition display and RUN button is orange<\/td><td class=\"column-2\">RUN is not activated<\/td><td class=\"column-3\">Press the RUN button<\/td>\n<\/tr>\n<tr class=\"row-3\">\n\t<td class=\"column-2\">Sample tube is not installed or is not properly seated<\/td><td class=\"column-3\">Install the sample tube correctly on the SIP<\/td>\n<\/tr>\n<tr class=\"row-4\">\n\t<td class=\"column-2\">Sample tube is cracked<\/td><td class=\"column-3\">Replace the sample tube<\/td>\n<\/tr>\n<tr class=\"row-5\">\n\t<td class=\"column-2\">Sheath container is not pressurized<\/td><td class=\"column-3\">1. Ensure that the sheath container lid and all connectors are securely seated.<br \/>\n2. Inspect the O-ring and replace it if necessary. Contact FMCF staff.<\/td>\n<\/tr>\n<tr class=\"row-6\">\n\t<td class=\"column-2\">Bal seal is worn<\/td><td class=\"column-3\">Replace the Bal seal. Contact FMCF staff<\/td>\n<\/tr>\n<tr class=\"row-7\">\n\t<td class=\"column-2\">Air leak at sheath container<\/td><td class=\"column-3\">Ensure that the sheath container lid and all connectors are securely seated<\/td>\n<\/tr>\n<tr class=\"row-8\">\n\t<td class=\"column-2\">Sheath container is empty<\/td><td class=\"column-3\">Fill the sheath container<\/td>\n<\/tr>\n<tr class=\"row-9\">\n\t<td class=\"column-2\">Air in sheath filter<\/td><td class=\"column-3\">Purge the filter<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<!-- #tablepress-33 from cache -->\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-798 troubleshooting \"><span class=\"entry-title rich-snippet-hidden\">No fluorescent signal<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T20:56:07+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-798\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-798\" href=\"#collapse-1-798\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">No fluorescent signal<\/div><\/a><\/h4><\/div><div id=\"collapse-1-798\" aria-labelledby=\"faq_1-798\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-14 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-14 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-14\">\n<table id=\"tablepress-34\" class=\"tablepress tablepress-id-34 tablepress-responsive tbody-has-connected-cells\">\n<thead>\n<tr class=\"row-1\">\n\t<th class=\"column-1\"><strong>Observation<\/strong><\/th><th class=\"column-2\"><strong>Possible causes<\/strong><\/th><th class=\"column-3\"><strong>Recommended solutions<\/strong><\/th>\n<\/tr>\n<\/thead>\n<tbody class=\"row-striping row-hover\">\n<tr class=\"row-2\">\n\t<td rowspan=\"3\" class=\"column-1\">No fluorescent signal<\/td><td class=\"column-2\">Incorrect fluorochrome assignment<\/td><td class=\"column-3\">Make sure the cytometer configuration in the software matches the optical filters in the cytometer<\/td>\n<\/tr>\n<tr class=\"row-3\">\n\t<td class=\"column-2\">Wrong filter is installed<\/td><td class=\"column-3\">Make sure the appropriate filter is installed for each fluorochrome<\/td>\n<\/tr>\n<tr class=\"row-4\">\n\t<td class=\"column-2\">Laser is not functioning<\/td><td class=\"column-3\">Verify the laser malfunction by changing the threshold to an alternative laser while running the appropriate sample. If unsuccessful, contact FMCF staff<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<!-- #tablepress-34 from cache -->\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-804 troubleshooting \"><span class=\"entry-title rich-snippet-hidden\">High event rate<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T20:56:13+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-804\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-804\" href=\"#collapse-1-804\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">High event rate<\/div><\/a><\/h4><\/div><div id=\"collapse-1-804\" aria-labelledby=\"faq_1-804\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-15 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-15 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-15\">\n<table id=\"tablepress-35\" class=\"tablepress tablepress-id-35 tablepress-responsive tbody-has-connected-cells\">\n<thead>\n<tr class=\"row-1\">\n\t<th class=\"column-1\"><strong>Observation<\/strong><\/th><th class=\"column-2\"><strong>Possible causes<\/strong><\/th><th class=\"column-3\"><strong>Recommended solutions<\/strong><\/th>\n<\/tr>\n<\/thead>\n<tbody class=\"row-striping row-hover\">\n<tr class=\"row-2\">\n\t<td rowspan=\"5\" class=\"column-1\">High event rate<\/td><td class=\"column-2\">Air bubble in the sheath filter or flow cell<\/td><td class=\"column-3\">Remove the air bubble from filter<\/td>\n<\/tr>\n<tr class=\"row-3\">\n\t<td class=\"column-2\">Sample is too concentrated<\/td><td class=\"column-3\">Dilute the sample<\/td>\n<\/tr>\n<tr class=\"row-4\">\n\t<td class=\"column-2\">Sample flow rate is set on HI<\/td><td class=\"column-3\">Set the sample flow rate to MED or LO<\/td>\n<\/tr>\n<tr class=\"row-5\">\n\t<td class=\"column-2\">Threshold level is too low<\/td><td class=\"column-3\">Increase the threshold level<\/td>\n<\/tr>\n<tr class=\"row-6\">\n\t<td class=\"column-2\">PMT voltage for the threshold parameter set too high<\/td><td class=\"column-3\">Set the PMT voltage lower for the threshold parameter<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<!-- #tablepress-35 from cache -->\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-806 troubleshooting \"><span class=\"entry-title rich-snippet-hidden\">Low event rate<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T20:56:21+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-806\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-806\" href=\"#collapse-1-806\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">Low event rate<\/div><\/a><\/h4><\/div><div id=\"collapse-1-806\" aria-labelledby=\"faq_1-806\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-16 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-16 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-16\">\n<table id=\"tablepress-36\" class=\"tablepress tablepress-id-36 tablepress-responsive tbody-has-connected-cells\">\n<thead>\n<tr class=\"row-1\">\n\t<th class=\"column-1\"><strong>Observation<\/strong><\/th><th class=\"column-2\"><strong>Possible causes<\/strong><\/th><th class=\"column-3\"><strong>Recommended solutions<\/strong><\/th>\n<\/tr>\n<\/thead>\n<tbody class=\"row-striping row-hover\">\n<tr class=\"row-2\">\n\t<td rowspan=\"5\" class=\"column-1\">Low event rate<\/td><td class=\"column-2\">Threshold level is too high<\/td><td class=\"column-3\">Lower the threshold level<\/td>\n<\/tr>\n<tr class=\"row-3\">\n\t<td class=\"column-2\">PMT voltage for the threshold parameter is set too low<\/td><td class=\"column-3\">Set the PMT voltage higher for the threshold parameter<\/td>\n<\/tr>\n<tr class=\"row-4\">\n\t<td class=\"column-2\">Sample is not adequately mixed<\/td><td class=\"column-3\">Mix the sample to suspend the cells<\/td>\n<\/tr>\n<tr class=\"row-5\">\n\t<td class=\"column-2\">Sample is too diluted<\/td><td class=\"column-3\">Concentrate the sample. If the flow rate setting is not critical to the application, set the flow rate switch to MED or HI<\/td>\n<\/tr>\n<tr class=\"row-6\">\n\t<td class=\"column-2\">Sample injection tube is clogged<\/td><td class=\"column-3\">Remove the sample tube to allow backflushing. If the event rate is still erratic, clean the sample injection tube<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<!-- #tablepress-36 from cache -->\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-808 troubleshooting \"><span class=\"entry-title rich-snippet-hidden\">Erratic event rate<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T20:56:28+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-808\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-808\" href=\"#collapse-1-808\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">Erratic event rate<\/div><\/a><\/h4><\/div><div id=\"collapse-1-808\" aria-labelledby=\"faq_1-808\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-17 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-17 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-17\">\n<table id=\"tablepress-37\" class=\"tablepress tablepress-id-37 tablepress-responsive tbody-has-connected-cells\">\n<thead>\n<tr class=\"row-1\">\n\t<th class=\"column-1\"><strong>Observation<\/strong><\/th><th class=\"column-2\"><strong>Possible causes<\/strong><\/th><th class=\"column-3\"><strong>Recommended solutions<\/strong><\/th>\n<\/tr>\n<\/thead>\n<tbody class=\"row-striping row-hover\">\n<tr class=\"row-2\">\n\t<td rowspan=\"5\" class=\"column-1\">Erratic event rate<\/td><td class=\"column-2\">Sample tube is cracked<\/td><td class=\"column-3\">Replace the sample tube<\/td>\n<\/tr>\n<tr class=\"row-3\">\n\t<td class=\"column-2\">Bal seal is worn<\/td><td class=\"column-3\">Replace the Bal seal. Contact FMCF staff<\/td>\n<\/tr>\n<tr class=\"row-4\">\n\t<td class=\"column-2\">Sample injection tube is clogged<\/td><td class=\"column-3\">Remove the sample tube to allow backflushing and prime the instrument several times. Contact FMCF staff if the issue is not resolved<\/td>\n<\/tr>\n<tr class=\"row-5\">\n\t<td class=\"column-2\">Contaminated sample<\/td><td class=\"column-3\">Prepare the specimen again. Ensure that the tube is clean<\/td>\n<\/tr>\n<tr class=\"row-6\">\n\t<td class=\"column-2\">Sheath filter is dirty<\/td><td class=\"column-3\">Replace the filter. Contact FMCF staff<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<!-- #tablepress-37 from cache -->\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-824 troubleshooting \"><span class=\"entry-title rich-snippet-hidden\">Distorted scatter parameters<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T20:56:34+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-824\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-824\" href=\"#collapse-1-824\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">Distorted scatter parameters<\/div><\/a><\/h4><\/div><div id=\"collapse-1-824\" aria-labelledby=\"faq_1-824\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-18 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-18 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-18\">\n<table id=\"tablepress-38\" class=\"tablepress tablepress-id-38 tablepress-responsive tbody-has-connected-cells\">\n<thead>\n<tr class=\"row-1\">\n\t<th class=\"column-1\"><strong>Observation<\/strong><\/th><th class=\"column-2\"><strong>Possible causes<\/strong><\/th><th class=\"column-3\"><strong>Recommended solutions<\/strong><\/th>\n<\/tr>\n<\/thead>\n<tbody class=\"row-striping row-hover\">\n<tr class=\"row-2\">\n\t<td rowspan=\"5\" class=\"column-1\">Distorted scatter parameters<\/td><td class=\"column-2\">Cytometer settings are improperly adjusted<\/td><td class=\"column-3\">Optimize the scatter parameters<\/td>\n<\/tr>\n<tr class=\"row-3\">\n\t<td class=\"column-2\">Air bubble in sheath filter or flow cell<\/td><td class=\"column-3\">Purge the air from the filter<\/td>\n<\/tr>\n<tr class=\"row-4\">\n\t<td class=\"column-2\">Flow cell is dirty<\/td><td class=\"column-3\">Perform the system flush procedure. Contact FMCF staff<\/td>\n<\/tr>\n<tr class=\"row-5\">\n\t<td class=\"column-2\">Air leak at sheath container<\/td><td class=\"column-3\">Ensure that the sheath container lid is tight and all connectors are secure<\/td>\n<\/tr>\n<tr class=\"row-6\">\n\t<td class=\"column-2\">Hypertonic buffers or fixative<\/td><td class=\"column-3\">Replace the buffers and fixative<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<!-- #tablepress-38 from cache -->\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-828 troubleshooting \"><span class=\"entry-title rich-snippet-hidden\">Excessive amount of debris in display<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T20:56:42+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-828\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-828\" href=\"#collapse-1-828\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">Excessive amount of debris in display<\/div><\/a><\/h4><\/div><div id=\"collapse-1-828\" aria-labelledby=\"faq_1-828\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-19 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-19 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-19\">\n<table id=\"tablepress-39\" class=\"tablepress tablepress-id-39 tablepress-responsive tbody-has-connected-cells\">\n<thead>\n<tr class=\"row-1\">\n\t<th class=\"column-1\"><strong>Observation<\/strong><\/th><th class=\"column-2\"><strong>Possible causes<\/strong><\/th><th class=\"column-3\"><strong>Recommended solutions<\/strong><\/th>\n<\/tr>\n<\/thead>\n<tbody class=\"row-striping row-hover\">\n<tr class=\"row-2\">\n\t<td rowspan=\"6\" class=\"column-1\">Excessive amount of debris in display<\/td><td class=\"column-2\">Threshold level is too low<\/td><td class=\"column-3\">Increase the threshold level<\/td>\n<\/tr>\n<tr class=\"row-3\">\n\t<td class=\"column-2\">Sheath filter is dirty<\/td><td class=\"column-3\">Replace the filter. Contact FMCF staff<\/td>\n<\/tr>\n<tr class=\"row-4\">\n\t<td class=\"column-2\">Flow cell is dirty<\/td><td class=\"column-3\">Prime the instrument several times. Contact FMCF staff<\/td>\n<\/tr>\n<tr class=\"row-5\">\n\t<td class=\"column-2\">Dead cells or debris in sample<\/td><td class=\"column-3\">Examine the sample under a microscope<\/td>\n<\/tr>\n<tr class=\"row-6\">\n\t<td class=\"column-2\">Sample is contaminated<\/td><td class=\"column-3\">Re-stain the sample, ensure tube is clean<\/td>\n<\/tr>\n<tr class=\"row-7\">\n\t<td class=\"column-2\">Stock sheath fluid is contaminated<\/td><td class=\"column-3\">Contact FMCF staff<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<!-- #tablepress-39 from cache -->\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-830 troubleshooting \"><span class=\"entry-title rich-snippet-hidden\">High CV<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T20:56:48+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-830\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-830\" href=\"#collapse-1-830\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">High CV<\/div><\/a><\/h4><\/div><div id=\"collapse-1-830\" aria-labelledby=\"faq_1-830\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-20 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-20 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-20\">\n<table id=\"tablepress-40\" class=\"tablepress tablepress-id-40 tablepress-responsive tbody-has-connected-cells\">\n<thead>\n<tr class=\"row-1\">\n\t<th class=\"column-1\"><strong>Observation<\/strong><\/th><th class=\"column-2\"><strong>Possible causes<\/strong><\/th><th class=\"column-3\"><strong>Recommended solutions<\/strong><\/th>\n<\/tr>\n<\/thead>\n<tbody class=\"row-striping row-hover\">\n<tr class=\"row-2\">\n\t<td rowspan=\"6\" class=\"column-1\">High CV<\/td><td class=\"column-2\">Air bubble in sheath filter or flow cell<\/td><td class=\"column-3\">Purge the filter<\/td>\n<\/tr>\n<tr class=\"row-3\">\n\t<td class=\"column-2\">Sample flow rate is set too high<\/td><td class=\"column-3\">Set the sample flow rate lower<\/td>\n<\/tr>\n<tr class=\"row-4\">\n\t<td class=\"column-2\">Air leak at sheath container<\/td><td class=\"column-3\">Ensure that the sheath container lid is tight and all connectors are secure<\/td>\n<\/tr>\n<tr class=\"row-5\">\n\t<td class=\"column-2\">Flow cell is dirty<\/td><td class=\"column-3\">Prime the instrument several times. Contact FMCF staff<\/td>\n<\/tr>\n<tr class=\"row-6\">\n\t<td class=\"column-2\">Poor sample preparation<\/td><td class=\"column-3\">Repeat sample preparation<\/td>\n<\/tr>\n<tr class=\"row-7\">\n\t<td class=\"column-2\">Sample not diluted in same fluid as sheath fluid<\/td><td class=\"column-3\">Dilute the sample in the same fluid as you are using for sheath<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<!-- #tablepress-40 from cache -->\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><div class=\"fusion-panel panel-default fusion-faq-post fusion-faq-post-833 troubleshooting \"><span class=\"entry-title rich-snippet-hidden\">Poor QC results<\/span><span class=\"vcard rich-snippet-hidden\"><span class=\"fn\"><a href=\"https:\/\/lab.research.sickkids.ca\/fcf\/author\/nsiddiqui\/\" title=\"Posts by nsiddiqui\" rel=\"author\">nsiddiqui<\/a><\/span><\/span><span class=\"updated rich-snippet-hidden\">2017-11-09T20:56:55+00:00<\/span><div class=\"panel-heading\"><h4 id=\"faq_1-833\" class=\"panel-title toggle\"><a data-toggle=\"collapse\" class=\"collapsed\" data-parent=\"#accordian-1\" data-target=\"#collapse-1-833\" href=\"#collapse-1-833\" aria-expanded=\"false\"><div class=\"fusion-toggle-icon-wrapper\"><div class=\"fusion-toggle-icon-wrapper-main\"><div class=\"fusion-toggle-icon-wrapper-sub\"><i class=\"fa-fusion-box active-icon awb-icon-minus\" aria-hidden=\"true\"><\/i><i class=\"fa-fusion-box inactive-icon awb-icon-plus\" aria-hidden=\"true\"><\/i><\/div><\/div><\/div><div class=\"fusion-toggle-heading\">Poor QC results<\/div><\/a><\/h4><\/div><div id=\"collapse-1-833\" aria-labelledby=\"faq_1-833\" class=\"panel-collapse collapse\"><div class=\"panel-body toggle-content post-content\"><div class=\"fusion-fullwidth fullwidth-box fusion-builder-row-1-21 nonhundred-percent-fullwidth non-hundred-percent-height-scrolling\" style=\"--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;\" ><div class=\"fusion-builder-row fusion-row\"><div class=\"fusion-layout-column fusion_builder_column fusion-builder-column-21 fusion_builder_column_1_1 1_1 fusion-one-full fusion-column-first fusion-column-last\" style=\"--awb-bg-size:cover;\"><div class=\"fusion-column-wrapper fusion-flex-column-wrapper-legacy\"><div class=\"fusion-text fusion-text-21\">\n<table id=\"tablepress-41\" class=\"tablepress tablepress-id-41 tablepress-responsive tbody-has-connected-cells\">\n<thead>\n<tr class=\"row-1\">\n\t<th class=\"column-1\"><strong>Observation<\/strong><\/th><th class=\"column-2\"><strong>Possible causes<\/strong><\/th><th class=\"column-3\"><strong>Recommended solutions<\/strong><\/th>\n<\/tr>\n<\/thead>\n<tbody class=\"row-striping row-hover\">\n<tr class=\"row-2\">\n\t<td rowspan=\"6\" class=\"column-1\">Poor QC results<\/td><td class=\"column-2\">Air bubble or debris in flow cell<\/td><td class=\"column-3\">Prime the fluidics system<\/td>\n<\/tr>\n<tr class=\"row-3\">\n\t<td class=\"column-2\">Old or contaminated QC particles<\/td><td class=\"column-3\">Make new QC samples and perform the quality control procedure again<\/td>\n<\/tr>\n<tr class=\"row-4\">\n\t<td class=\"column-2\">Sample not diluted in same fluid as sheath fluid<\/td><td class=\"column-3\">Dilute the sample in the same fluid as you are using for sheath<\/td>\n<\/tr>\n<tr class=\"row-5\">\n\t<td class=\"column-2\">Laser not warmed up<\/td><td class=\"column-3\">Wait the recommended amount of time for the laser to warm up.<br \/>\n- 30 min for the 488-nm (blue)<br \/>\n- 30 min for the 355-nm (UV)<br \/>\n- 15 min for the 405-nm (violet)<br \/>\n- 20 min for the 633-nm (red)<\/td>\n<\/tr>\n<tr class=\"row-6\">\n\t<td class=\"column-2\">Laser not functioning<\/td><td class=\"column-3\">Contact FMCF staff<\/td>\n<\/tr>\n<tr class=\"row-7\">\n\t<td class=\"column-2\">Optical alignment problem<\/td><td class=\"column-3\">Contact FMCF staff<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<!-- #tablepress-41 from cache -->\n<\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n<\/div><\/div><\/div><\/div><\/div><\/div><\/div><div class=\"fusion-clearfix\"><\/div><\/div><\/div><\/div><\/div>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":2,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-733","page","type-page","status-publish","hentry"],"yoast_head":"<!-- This site is optimized with the Yoast SEO Premium plugin v27.0 (Yoast SEO v27.0) - 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