Sample Preparation for Cell Sorting

Sample Properties

Cells (if <40 μm) in all samples and controls must be filtered through a <40 μm nylon mesh filter to prevent clogging of the instrument right before they are brought into the sorting room or right before they are introduced into the cell sorter even if the cells have been filtered earlier before staining in the user’s lab.

Samples can be in any of the following tubes: 12 x 75mm, 15 mL, or 1.5 mL .

The concentration of lymphocyte-like cells can be up to 5 x 107 cells/mL.  

For a multicolor experiment, the user must bring an unstained control tube and a single-color stained samples (ideally >5% positive) to use for compensation.  The user should consult Core staff for the controls required for the instrument set up if there is any confusion.

Users need to bring collection container(s) which can be any of the following options:

  1. Tubes: 12 x 75mm, 15 mL, or 1.5-2 mL.  For 4-way sorting, only 12 x 75mm or 1.5-2mL tubes can be used.
  2. Plates: 6-,12-,24-,48-, 96- (including PCR plate), 384-well, or custom size
  3. Trays/dishes or slides

The collection container should be pre-filled with the desired volume of collection medium. 

 

If this is a new sort:

  1. Please bring extra sample buffer in case sample dilution is required.
  2. Please bring extra collection tubes/media, especially if a large number of cells is being collected and/or the sort requires a larger nozzle size.  (The droplet of sheath fluid (PBS) around each cell will add volume to your collected sample.)

Please comment on any other special handling requirements for your samples, i.e. light sensitivity, tendency to clump, keep on ice, etc. The more we know about your experiment, the better the results.

 

Safety Concerns

No radioactive materials are allowed in the facility.

Unfixed samples known to contain BSL-3 pathogens or unfixed samples recommended to sort at BSL3 will not be accepted. The Operational Director will make the decision concerning sorting of other known infectious agents on an individual basis in consultation with the EH&S Biosafety Officer when necessary.

Criteria for samples containing potential bio-hazards, such as unfixed human, non-human primate, virally-transduced or infected cells:

  1. Sample must be contained in a leak-proof container with a secure lid and be clearly labeled with a sample identifier and a biohazard symbol, if needed.
  2. Sample must be clump free and in an appropriate tube for sorting (12 x 75 snap cap tube or 15 mL tube with cap).
  3. Sample must not contaminate the outside of the tube.

Please indicate biosafety concerns when scheduling the sort. 

 

Viability Concerns

Sorting can be very stressful to cells.  By far the biggest stressors are the pressure changes experienced by the cell as its droplet goes through the nozzle.  Larger diameter nozzles sort more gently but samples must be run more slowly due to the lower pressure.  Thus there is a practical tradeoff between the time spent in the sorter (and outside of growth media/warmth) and the pressures required for an efficient sort.

FACS Aria sorters are designed and optimized for lymphocytes and other smaller/heartier cells.  Coming soon (Summer 2022) will be a BigFoot spectral sorter, which has a 150 µm nozzle available to sort larger cell types.  If you have larger cells and have had viability issues in the past, you may want to try your cells again on the BigFoot.

Other things that can be done to preserve cell viability/sort more efficiently

  • Liquid in the collection tube
    • Cells that hit a dry surface will almost always die.  Eventually enough dead cells and sheath buffer (PBS) will build up to give the later cells a chance to survive, but why lose cells unnecessarily???
  • Serum
    • Many protocols recommend a lower serum concentration during sorting, and compensate with a higher serum concentration in the collection media.
  • pH
    • Most cell media buffers are bicarbonate based, and will tend to be alkaline outside the incubator’s 5% CO2 atmosphere.  If your sorts and benchwork take a long time outside the hood, consider adding sterile HEPES buffer to your media.  Especially if your red media looks a little more pink/purple.
  • Temperature
    • Some cells (not all) will benefit from being kept cool until they can be returned to growth media and the incubator.
      • Our sorters have optional cooling jackets for sample collection.  Simply request one in the comment section when you schedule the sort.
      • If you have several samples or they run slowly, leave an ice bucket with us to keep your waiting samples cool.
  • Sample enrichment
    • Consider using a method such as magnetic beads to pull out cell populations you are NOT interested in, so that your cells of interest comprise a larger percentage of the remaining sample.  The sort will run faster, reducing cell stress and sort costs.
  • Serum Compatible Live/Dead dye
    • This makes sure the cells only live cells are sorted.
    • We have observed that larger cells do take up some live/dead dye, although not as much as dead cells.  Sometimes with larger cells you’ll want to collect the dim cells along with the negative cells, and exclude only the bright dead ones.
      • You can always do a control wherein you take a population of living cells, heat kill ½ of them, recombine and stain with your favorite viability dye.  This should give you a clear indication where to gate.
  • Azide
    • Some commercial flow cytometry buffers contain sodium azide.  Make sure there is no azide in any buffers where the cells are expected to live.