{"id":1804,"date":"2021-09-16T15:10:23","date_gmt":"2021-09-16T15:10:23","guid":{"rendered":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/?page_id=1804"},"modified":"2021-09-16T15:12:19","modified_gmt":"2021-09-16T15:12:19","slug":"conditioned-media-protocol","status":"publish","type":"page","link":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/conditioned-media-protocol\/","title":{"rendered":"Conditioned Media Protocol"},"content":{"rendered":"

PROTOCOL<\/strong><\/span><\/h1>\n

Conditioned Media Sample Preparation<\/strong><\/span><\/h1>\n
    \n
  1. Grow cells of interest at relatively high density (110, 000 cells\/cm2). I usually use 5 x 10-cm dishes to yield ~50 ml of conditioned media.<\/span><\/li>\n
  2. Add culture media to an identical number of culture dishes treated identically but do not add any cells to these dishes. This media will serve as the negative control.<\/span><\/li>\n
  3. Change the media to serum-free growth media and allow cells to condition the media for 24-48 hours. Change media on the control plates as well.<\/span><\/li>\n
  4. Collect the media from all of the plates, centrifuge at 1000g for 10 min and carefully remove the supernatant, leaving a small quantity of media behind. The point of this step is to make sure there are no cells in the media. Combine all of the media from each of the 5 plates to yield 50 ml of conditioned media and 50 ml of control media.<\/span><\/li>\n
  5. Pre-wash an Amicon Ultra-15 centrifugal filter (3 kDa MWCO) with 50 mM ammonium bicarbonate pH 8 for 10 min at 3500 rpm.<\/span><\/li>\n
  6. Add 15 ml of the conditioned media to the Amcion and spin at 3500 rpm for 30 minutes. Repeat these intervals until the volume is reduced to 1-2 ml.<\/span><\/li>\n
  7. Fill Amicon with 50 mM ammonium bicarbonate pH 8.<\/span><\/li>\n
  8. Spin Amicon at 3500 rpm for 30 min intervals until the volume reduces to ~1-2 ml.<\/span><\/li>\n
  9. Repeat steps 7 and 8 until the final volume is ~250 ul and there is no pink colour (from the phenol red) that is detectable by eye. Usually takes at least 5 repeats.<\/span><\/li>\n
  10. Determine protein concentration.<\/span><\/li>\n
  11. Lyophilize solution until dry.<\/span><\/li>\n<\/ol>\n

     <\/p>\n

    Download and save a PDF copy of the Conditioned Media Protocol.\u00a0<\/strong><\/em><\/span><\/a><\/p>\n","protected":false},"excerpt":{"rendered":"

    PROTOCOL Conditioned Media Sample Preparation Grow cells of interest at relatively high density (110, 000 cells\/cm2). I usually use 5 x 10-cm dishes to yield ~50 ml of conditioned media. Add culture media to an identical number of culture dishes treated identically but do not add any cells to these dishes. This media will serve…<\/p>\n","protected":false},"author":205,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-1804","page","type-page","status-publish","hentry","description-off"],"yoast_head":"\nConditioned Media Protocol - SPARC BioCentre Molecular Analysis<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/conditioned-media-protocol\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Conditioned Media Protocol\" \/>\n<meta property=\"og:description\" content=\"PROTOCOL Conditioned Media Sample Preparation Grow cells of interest at relatively high density (110, 000 cells\/cm2). 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