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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.