FAQ2019-08-30T15:27:18+00:00
Droplet containment vacuum not functioning2017-11-09T20:55:31+00:00
ObservationPossible causesRecommended solutions
Droplet containment vacuum not functioningWorn O-ring in retainerReplace the O-ring. Contact FMCF staff.
Outer sleeve is not seated in the retainer1. Loosen the retainer
2. Push the outer sleeve up into the retainer until seated.
3. Tighten the retainer
Outer sleeve is not on the sample injection tubeReplace the outer sleeve.
1. Loosen the retainer.
2. Slide the outer sleeve over the sample injection tube until it is seated.
3. Tighten the retainer.
Waste line is pinched, preventing proper aspirationCheck the waste line.
Waste tank is fullEmpty the waste tank.
How many cells do I need to bring to the sorter?2018-01-23T19:20:54+00:00

This is the most commonly asked question. To answer it we need to know the following information:

  1. What is the approximate percentage of the population(s) you wish to sort?
  2. How many total cells do you want back?
  3. Do you want stringent purification or enrichment?
  4. How fragile or large are your cells?

All of these parameters influence the yield and purity of cell sorting. Here is an example: Let’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.

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).

Sample tube not fitting on SIP2018-07-27T15:46:33+00:00
ObservationPossible causesRecommended solutions
Sample tube not fitting on SIPSample tube other than BD Falcon tubes usedUse BD Falcon 12 x 75-mm sample tubes.
Tubes must be polystyrene NOT polypropylene
Worn Bal sealReplace the Bal seal. Contact FMCF staff
How long will it take to sort?2017-11-09T14:58:29+00:00

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).

For example, on the MoFlo cell sorter, we use the 100µm 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 µm nozzle at 30psi, or at 9,000-10,000/second with the 70 µm nozzle at 60psi.

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.

Do I need to filter my cells?2017-11-09T14:57:55+00:00

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.

Rapid sample aspiration2017-11-09T20:55:46+00:00
ObservationPossible causesRecommended solutions
Rapid sample aspirationSupport arm is to the sidePlace the support arm under the sample tube
Droplet containment module is failingCall your service representative. Contact FMCF staff
What do I re-suspend my cells in for sorting?2017-11-09T14:58:35+00:00

Proper sample preparation is absolutely essential for obtaining good purity and yields. 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) calcium and magnesium free containing 10 mM HEPES, pH 7.2).

Most cells benefit from having some protein in the media, such as 1-2% FCS, 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.

For sticky cells:  Increase the concentration of the EDTA to 5mM and use FBS 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.

For adherent cells:  To detach adherent cells, trypsin (or other detachment buffer) is often quenched with media or buffer containing serum.  This reintroduces cations that can promote aggregation. Use FBS 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 that can be used to generate single cell suspensions.

NOTE: Accutase can alter some cell surface epitopes; the effect will need to be determined empirically for the epitopes being evaluated.

For samples with a high percentage of dead cells:  DNA from dead cells can cause cell clumping. DNAse I in the presence of magnesium chloride will help reduce cellular aggregation.

  1. Treat cells for 15 to 30 minutes in a solution of 100 µg/mL DNAse and 5 mM MgCl2 in HBSS at room temperature.
  2. Wash the cells once in HBSS with 5 mM MgCl2.
  3. Gently resuspend the cells in HBSS with 5 mM MgCl2 and add 25-50 µg/mL DNAse (as a maintenance dose) prior to and during the sort.

Note: DNAse I requires at least 1 mM magnesium to work effectively, although 5mM is optimal. It is important to minimize the presence of dead cells during this procedure, since actin released from dead cells irreversibly inhibits DNAse I.

What controls do I need to bring?2018-07-27T15:46:13+00:00

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.

Please refer to the table below to see an example of a typical sort experiment with three colours.

Control typeSample NameMarker +
fluorochrome #1
Marker +
fluorochrome #2
Marker +
fluorochrome #3
Unstained ControlUnstained---
Single colour compensation controlsCD3 - FITCCD3 - FITC--
CD4 - PE-CD4 - PE-
CD8 - APC-CD8 - APC
FMO ControlFMO FITC-CD4 - PECD8 - APC

Fluorophores excited by the Yellow/Green laser
If 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.

Viability Dyes
We 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.

What are FACS Tubes and where do I get them?2017-11-09T20:55:03+00:00

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).

What kind of tubes or plates should the cells be sorted into?2017-11-09T14:59:05+00:00

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.

No events in acquisition display and RUN button is green2017-11-09T20:55:54+00:00
ObservationPossible causesRecommended solutions
No events in acquisition display and RUN button is greenThreshold is not set to the correct parameter (usually FSC)Set the threshold to the correct parameter for your application
Threshold level is too highLower the threshold level
PMT voltage for threshold parameter is set too lowSet the PMT voltage higher for the threshold parameter
Gating issueEnsure gating strategy is appropriate
Air in the sheath filterPurge the filter
Sheath and air line not connected to the sheath tankEnsure sheath and air line are connected to the sheath tank
O-ring is not in position in the sheath tankReposition O-ring in sheath tank
Cracked tube/ Bal seal (tube to SIP connection) problemReplace tube
No sample in the tubeAdd sample to the tube or install a new sample tube
Sample is not mixed properlyMix the sample to suspend cells
Waste tank is fullEmpty the waste tank
PMT voltages set too low or too high for display parameterReset the PMT voltages
Too few events are displayedIncrease the number of events to display
Sample injection tube is cloggedRemove the sample tube to allow backflushing and prime the instrument several times. Clean instrument. Contact FMCF staff if issue is not resolved
Laser is not warmed upWait the recommended amount of time for the laser to warm up.
- 30 min for the 488-nm (blue)
- 30 min for the 355-nm (UV)
- 15 min for the 405-nm (violet)
- 20 min for the 633-nm (red)
Laser delay is set incorrectlyContact FMCF staff
Laser is not functioningVerify the malfunction by changing the threshold to an alternative laser while running the appropriate sample. If unsuccessful, contact FMCF staff
What collection medium should the cells be sorted into?2017-11-09T14:59:16+00:00

We recommend 100% serum or HEPES buffered HBSS.

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’s best to start with it on the acid side of neutral.

No events in acquisition display and RUN button is orange2017-11-09T20:56:01+00:00
ObservationPossible causesRecommended solutions
No events in acquisition display and RUN button is orangeRUN is not activatedPress the RUN button
Sample tube is not installed or is not properly seatedInstall the sample tube correctly on the SIP
Sample tube is crackedReplace the sample tube
Sheath container is not pressurized1. Ensure that the sheath container lid and all connectors are securely seated.
2. Inspect the O-ring and replace it if necessary. Contact FMCF staff.
Bal seal is wornReplace the Bal seal. Contact FMCF staff
Air leak at sheath containerEnsure that the sheath container lid and all connectors are securely seated
Sheath container is emptyFill the sheath container
Air in sheath filterPurge the filter
No fluorescent signal2017-11-09T20:56:07+00:00
ObservationPossible causesRecommended solutions
No fluorescent signalIncorrect fluorochrome assignmentMake sure the cytometer configuration in the software matches the optical filters in the cytometer
Wrong filter is installedMake sure the appropriate filter is installed for each fluorochrome
Laser is not functioningVerify the laser malfunction by changing the threshold to an alternative laser while running the appropriate sample. If unsuccessful, contact FMCF staff
High event rate2017-11-09T20:56:13+00:00
ObservationPossible causesRecommended solutions
High event rateAir bubble in the sheath filter or flow cellRemove the air bubble from filter
Sample is too concentratedDilute the sample
Sample flow rate is set on HISet the sample flow rate to MED or LO
Threshold level is too lowIncrease the threshold level
PMT voltage for the threshold parameter set too highSet the PMT voltage lower for the threshold parameter
Low event rate2017-11-09T20:56:21+00:00
ObservationPossible causesRecommended solutions
Low event rateThreshold level is too highLower the threshold level
PMT voltage for the threshold parameter is set too lowSet the PMT voltage higher for the threshold parameter
Sample is not adequately mixedMix the sample to suspend the cells
Sample is too dilutedConcentrate the sample. If the flow rate setting is not critical to the application, set the flow rate switch to MED or HI
Sample injection tube is cloggedRemove the sample tube to allow backflushing. If the event rate is still erratic, clean the sample injection tube
Erratic event rate2017-11-09T20:56:28+00:00
ObservationPossible causesRecommended solutions
Erratic event rateSample tube is crackedReplace the sample tube
Bal seal is wornReplace the Bal seal. Contact FMCF staff
Sample injection tube is cloggedRemove the sample tube to allow backflushing and prime the instrument several times. Contact FMCF staff if the issue is not resolved
Contaminated samplePrepare the specimen again. Ensure that the tube is clean
Sheath filter is dirtyReplace the filter. Contact FMCF staff
Distorted scatter parameters2017-11-09T20:56:34+00:00
ObservationPossible causesRecommended solutions
Distorted scatter parametersCytometer settings are improperly adjustedOptimize the scatter parameters
Air bubble in sheath filter or flow cellPurge the air from the filter
Flow cell is dirtyPerform the system flush procedure. Contact FMCF staff
Air leak at sheath containerEnsure that the sheath container lid is tight and all connectors are secure
Hypertonic buffers or fixativeReplace the buffers and fixative
Excessive amount of debris in display2017-11-09T20:56:42+00:00
ObservationPossible causesRecommended solutions
Excessive amount of debris in displayThreshold level is too lowIncrease the threshold level
Sheath filter is dirtyReplace the filter. Contact FMCF staff
Flow cell is dirtyPrime the instrument several times. Contact FMCF staff
Dead cells or debris in sampleExamine the sample under a microscope
Sample is contaminatedRe-stain the sample, ensure tube is clean
Stock sheath fluid is contaminatedContact FMCF staff
High CV2017-11-09T20:56:48+00:00
ObservationPossible causesRecommended solutions
High CVAir bubble in sheath filter or flow cellPurge the filter
Sample flow rate is set too highSet the sample flow rate lower
Air leak at sheath containerEnsure that the sheath container lid is tight and all connectors are secure
Flow cell is dirtyPrime the instrument several times. Contact FMCF staff
Poor sample preparationRepeat sample preparation
Sample not diluted in same fluid as sheath fluidDilute the sample in the same fluid as you are using for sheath
Poor QC results2017-11-09T20:56:55+00:00
ObservationPossible causesRecommended solutions
Poor QC resultsAir bubble or debris in flow cellPrime the fluidics system
Old or contaminated QC particlesMake new QC samples and perform the quality control procedure again
Sample not diluted in same fluid as sheath fluidDilute the sample in the same fluid as you are using for sheath
Laser not warmed upWait the recommended amount of time for the laser to warm up.
- 30 min for the 488-nm (blue)
- 30 min for the 355-nm (UV)
- 15 min for the 405-nm (violet)
- 20 min for the 633-nm (red)
Laser not functioningContact FMCF staff
Optical alignment problemContact FMCF staff