Pre-sort prep... clumps, tubes, cell concentration, controls, temperature
Call the lab and talk to the sort operator to discuss your sort (you will be grilled). What kind of cells are you sorting? How many cells do you need? How much time is needed? Which fluorochromes? Fill out the pre-sort form. Email it or bring it with you for the discussion.
Pre-sort cells must be in a single-cell suspension. Clumps cause instability in the flow tip or can clog, causing frazzlement of the sorter operator. Stream turbulence will cause lower purities in your sorted tubes. We recommend a concentration of between 5 and 40 million per ml. The prep can be suspended in the cell's happiest buffer to keep them alive during the sort. Bring extra buffer for dilution of a too concentrated sample. (It's always easier to dilute rather than concentrate a sample.)
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|
|20,000||10 x 106||1||180|
Or calculate likely rates for yourself using the tools at http://facs.scripps.edu/recovery.html.
All cell samples must be filtered through 40 um nylon mesh to remove clumps of tissue, etc. We have nylon mesh filters in the lab. These can be sterilized by autoclaving. Pour your sample through the mesh prior to sorting. Rinsing with clear buffer will dilute your cell concentration, but help to avoid loss due to clogging. Filtering often causes loss of cells, but is unavoidable for a trouble-free sort. To determine how many cells are lost during a sort, count the cells after all antibody staining preparation and filtering, not just at the beginning of your prep.
Important Point: If filtering through mesh isn’t possible, be sure to call the lab and cancel your sort appointment.
Bring your pre-sort single cell suspension in a Falcon #352063 12 x 75mm tube. This is a specific requirement—other tubes will not fit the sample head.
Always, always, always, every time, bring an unstained control, along with single color preps for each different fluorochrome you use in your sorting sample. Bring them in Falcon tubes (see above). The operator may refuse to sort if proper controls are not provided.
Sorted samples can be collected into 1.5 microfuge tubes, 12x75 test tubes, or 15 or 50 ml conical centrifuge tubes. Bring the tubes of choice with you, containing a modicum of "happy" buffer to cushion the sorted cells. Always bring extra tubes. A clog will result in completely restarting the sort, discarding the contaminated collection tube. If sorting onto a plate (6, 12, 24, 96 well) only one population can be sorted at a time.
It is possible to control the temperature of pre and post sort tubes. Please let us know in advance if this is necessary.
Sterile sorts are indeed possible. We will run 70% EtOH or bleach through the sheath and sample tubing, use sterile sheath fluid and sterilize the sort chamber with UV light. You must remember that all preparation of the sample must be done sterility and brought to the lab in a sterile fashion and should include antibiotics in the media—both the sample and collection tubes. Control tubes should also be sterile preps. Collection tubes must be sterile with sterile media containing antibiotics. We cannot control what you bring into the lab. Remember most common bacterial contamination is caused by handling, not from the air.