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Sorting Quickstart
Thinking about sorting?? Here are a few details on how to prepare cells and estimate sort rates.
Why sort?
The researcher can recover two different populations and discard the remainder of the cells. Any parameter, or combination of parameters, can be used as a sort criterion. Typical choices are: FALS and SS for size and complexity, coupled with fluorescent tags for specific markers. Potential candidate cells for sorting should be "pre-tested" on the Coulter XL-MCL cytometer long before sorting enters the conversation.
Cell preparation for sorting
Prepare a single-cell suspension at approximately 5 to 40 x 106 cells/ml in 12x75mm culture tubes (Falcon #352063). Be sure cells will not clump and will tolerate sitting at room temperature for the duration of the sort. Cells must withstand dilution into sheath buffer (you may prepare your own specialty buffer if they can not). Know how many cells you need and adjust the sort time accordingly. Bring several tubes (culture tubes or 15ml centrifuge tubes) with media for collection vessels. For autoclone sorting, bring multiwell plates with 100ul of media in wells. A CO2 incubator is available for short-term storage. Preparation details.
Sort rates
10,000 to 20,000 particles per second is a reasonable estimate for expected sorting rate. Remember that your sorted population depends on the percentage of "good" cells in the start population, as this table illustrates:
| Sort rate (particles/s) | Start concentration (particles/ml) | Starting % | Time to sort 106 cells (minutes) |
|---|---|---|---|
| 10,000 | 5 x 106 | 1 | 240 |
| 10 | 35 | ||
| 50 | 10 | ||
| 20,000 | 10 x 106 | 1 | 180 |
| 10 | 20 | ||
| 50 | 5 |
Or calculate likely rates for yourself using the tools at http://facs.scripps.edu/recovery.html.