Sample Guidelines & Protocols

CELL SORTING

• Single cell suspensions: cell sorting works best only if cells are in single cell suspension at the ideal cell concentration.

70µm nozzle, Lymphocytes and Small Cells (>14 µm), 0.75-1.2 x 107 cells/mL

85 µm nozzle, Activated Lymphocytes, Stem Cells, Monocytes, Dendritic Cells, 0.5-0.75 x 107 cells/mL

100 µm nozzle, Cell Lines, Macrophages, Stem Cells, Disaggregated Solid Tissue, 0.5-0.75 x 107 cells/mL

130µm nozzle, Fibroblasts, 0.2-0.5 x 107 cells/mL.  Sorting with this size nozzle is the sorter pressure regulator’s lower boundary and      can give rise to random and uneven droplet formation

• Filter samples just prior to sort using a 35-70µm cell strainer even if you filtered before staining
• To maintain cell viability outside of an incubator, keep samples on ice or at 2-8°C unless directed by a specific protocol
• Increase the purity and efficiency when sorting rare populations (less than 1%), or dim cells by cell enrichment/depletion. Cells can be enriched by density gradient centrifugation, magnetic bead-based technology, or by double sorting first for yield and then purity.
• Always use a red blood cell lysis buffer if the sample is suspected to contain unwanted RBCs.
• Consider removing dead cells by using a ficoll gradient or identifying them with the use of a viability dye.
• Controls: Controls are essential for setting voltages, calculating concentrations, and gating for the population of interest
  • • Negative/Unstained Control: Cells which were mock-transfected or not stained
    • Single Stained (Compensation) Controls: Single antibody-stained beads or cells for every fluorophore
    • Fluorescence Minus One (FMO) Controls: Cells which are stained with all fluorophores, minus one of them
• Sorting buffer:
  • • 1 x PBS (Ca/Mg++ free)
    • 2% dialyzed FBS (heat inactivated) or 0.5-2%BSA
    • 25mM HEPES p.H. 7.0
    • Optional: HBSS without Phenol Red may be substituted for 1 x PBS
    • Optional: Add a dead cell discriminator such as PI or Sytox to exclude dead cells
    • Optional: Add EDTA to 1-5mM to reduce cell aggregation
    • Optional: Pretreat sample with 10U/M DNase I if large number of dead cells are present (Omit EDTA in buffers containing DNase)
    • Cell culture medium and buffers containing Phenol Red or high protein concentrations are not recommended due to issues with drop in pH, increased fluorescence background, or creation of a refractive bias
• Collection Media:
  • • Culture media containing FBS in sufficient concentrations for growth, antibiotics, and 10-25mM HEPES p.H. 7.0
    • 1 x PBS with 10-50% FBS, antibiotics, and 10-25mM HEPES p.H. 7.0
    • Cell lysis buffer such as Trizol LS or Qiagen RLT buffer