Flow Cytometry Protocols: Sorting Checklist

Sorting is by appointment only. Telephone (858) 822-0407 to set an appointment or for more information. You may need to use voicemail, but do not e-mail.

Sample

  • Provide tubes pre-filled with 4 ml of the sample buffer (no cells) for rinsing.
  • Suspend cells in PBS or HBSS with 5% (2 - 10%) BSA at up to 3 X 107 per ml in Falcon ® [35]2063 12 X 75 mm or [35]2097 17 X 120 polypropylene tubes.
    • [35]2054 or [35]2058 12 X 75 mm polystyrene tubes can also be used.
    • Serum as the carrier protein is NOT recommended, as it adds to the background fluorescence signal.
  • Particle concentration effects sort speed. About 1 ml/hr of sample can be processed:
    • 7 X 106 TOTAL particles per ml is about 25 million per hour
      • Use the highest concentration possible while maintaining a single cell suspension
        • 20 - 100 million cells/ml
      • We can dilute sample with your provided buffer at the sorter, but can't concentrate them.
  • Filter cells through
    • Falcon® Cell Strainers ([35]2340) for large numbers of cells
    • Cell-Strainer Caps ([35]2235) can also be useful
  • Some cell types may need to be treated with DNAse to remove dead cell clumps and may also need to be suspended in DNAse buffer to maintain a single cell suspension. See DNAse Protocol or use Accumax from Innovative Cell Technologies.
    • An alternate solution to prevent aggregates is 1 mM EDTA.
      • NOT compatible with DNAse!

Bulk Collection

  • Sorted cells should NOT be collected into empty tubes.
    • 100% serum is recommended for bulk sorting.
    • Tubes should be kept cold & in the dark.
  • FOUR populations can simultaneously be sorted into:
    • 12 x 75 mm tube (Falcon® [35]2063 is the best)
      • Use 1 ml serum per 12 X 75 mm tube
      • Holds about 0.5 million sorted cells
    • 1.5 ml Eppendorf tubes
      • Cut the hinge!
    • 1.2 ml microtiter tubes (Fisher #02-681-383)
  • TWO populations can be collected into tubes above or larger tubes
      • eg, 17 X 120 mm tube Falcon® [35]2097)
      • Use 1 ml serum per 17 X 120 mm tube
        • Will hold about 2 million sorted cells

Automated Cell Deposition Unit (ACDU)

  • Single cells can be sorted at 1 - 50,000 particles:
    • Onto a slide or into microtiter wells (6,12,24,48,96 or 384 wells per plate)
      • Plates should be pre-filled (100 microliters conditioned media in 96 well plates) and pre-gassed.
        • Keep in a sealed sandwich bag or keep a tiny chip of CO2 in the plate's container (large box) to keep bicarbonate-buffered media's pH correct (pink media is not good).
        • Or use 50% serum in PBS and change buffer when you return to your lab.
      • Can also directly sort into PCR extraction buffer or other non-culture buffer (i.e. serum)
    Cell cloning works best in phenol red-free media and/or on feeder cells
  • 1,000 cells = 3.5 microliters sheath (PBS), of which only approximately 1% is the original sample solution

Sort Estimator

Example to estimate THEORETICAL yield (Y):

Yield = R*P/100*EXP(-R*n*T)

where T = 3.7*[SQRT(p)/4.5*(d)/106] = 69,490 Hz

and

R = 6,800 cells per second
P = 1 percent positive
d = 70 micron nozzle
p = 35 psi
n = 1.5 drop packet

or

58 cells per second (211,380 per hour), 86% recovery.

This does NOT count sort aborts.