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Caltech
Flow Cytometry
and Cell Sorting Facility
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Common Recommendations

These are some common recommendations that we often share with users during their consultations. While these are broadly applicable for the most part, please keep in mind that your sample or assay may have different requirements. Please always feel free to reach out to [email protected] with any questions.

Shelley's Buffer – HBSS:

Ideal for sorting some cell types. Some ingredients may be optional.

  • HBSS without phenol red, Ca2+ or Mg2+
  • 10mM HEPES buffer (be careful to use the cell culture variety and not just a chemical buffer)
  • DNAse1 (50ug/ml) from Roche (added the same day as the sort, to minimize aggregation due to cell death)
  • 1mM MgCl2
  • BSA Fraction V (2.5mg/ml)
    • Be sure to check your pH (BSA is acidic)

Useful Links:

Trypsin inhibitor

Falcon tube cap strainers

Celltrics strainers (50um)

5ml Polypropylene Tubes

Keep In Mind:

  • Unstained controls are important for setting up the instrument. Please bring them if possible. Without controls, we cannot guarantee that what looks positive is actually positive, etc!
  • For immunophenotyping - match bright flourophores with rare targets, and vice versa. FluoroFinder is a good resource for panel building.If you are viewing live cells, add a Live/Dead / Viability dye.
  • Bring any supplies you must have, especially if you require a sterile environment to work with your sample.
  • If you use an enzyme, adding an inhibitor is recommended. Otherwise, the enzyme may continue to effect your sample, even with after being washed.
  • It is better to have a sample that is too concentrated than too dilute, as you can always further dilute your sample.
  • Bring extra buffer to add to your collection tubes or sample.

Tips for Single Cell Suspension:

  • Keep your sample on ice.
  • Strain your sample through at least a 50 um filter as close to your start time as possible.
  • Add protein (~1-2% BSA or FBS) to your buffer to prevent clumping and/or non-specific binding.
  • DNase can be added to your sample to prevent clumping caused by dead cells releasing DNA into solution.
  • EDTA can be added to your sample to prevent clumping caused by cations (ex. Mg++). Note: chelation of cations will prevent DNase from working.

Analyzing Tips:

  • An ideal sample concentration is 1-5 million cells per mL.
  • Keep the speed on the analyzer set to low for tighter data peaks.
  • If you want to run more events per second make your samples more concentrated instead of adjusting the speed on the machine.

Sorting Tips:

  • An ideal sample concentration is 10-20 million cells per mL.
  • Bring extra collection tubes.
  • Always have your downstream in mind. For example, if you are sorting for 10X analysis, certain dyes may be off limits.