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.
- 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 ® 2063 12 X 75 mm or 2097 17 X 120 polypropylene tubes.
- 2054 or 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 (2340) for large numbers of cells
- Cell-Strainer Caps (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!
- 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® 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
- 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® 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:
Cell cloning works best in phenol red-free media and/or on feeder cells
- 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)
- 1,000 cells = 3.5 microliters sheath (PBS), of which only approximately 1% is the original sample solution
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
R = 6,800 cells per second
P = 1 percent positive
d = 70 micron nozzle
p = 35 psi
n = 1.5 drop packet
58 cells per second (211,380 per hour), 86% recovery.
This does NOT count sort aborts.