Preparing Cells for Sorting Protocol
Before any sort can be scheduled, we expect that you have some documented data that shows the levels of expression of the cells that are to be sorted. If there is none, we expect you to take a look at your cells on the benchtop cytometers so that we can help you calculate the number of cells you need for the sort and the length of time you will require.
If cells are to be sorted live, resuspend them in your culture medium that contains HEPES buffer. This extra buffering will protect the cells from any pH changes they might undergo during pressurization or charging of droplets during the sort process. The protein in the culture medium (often fetal calf serum) will help prevent the cells from sticking together.
If cells are fixed, resuspend them in buffered saline that contains some protein. It can be BSA (.1%) or fetal calf serum (1%).
For sorts done at lower pressures, resuspend cells at 10 X 106 cells/ml as described, or if there are less than 10 X 106 cells resuspend the cells in one ml. Unless cells are susceptible to cold, keep the sample on ice.
For sorts done at higher pressures, resuspend cells at a concentration of 15-20 X 106 cells/ml.
Please bring some extra culture medium with you to dilute your sample, if necessary.
If you are planning to isolate RNA from your sorted cells, we ask that you bring a liter of DEPC (diethethyl pyrocarbonate)-treated H2O that has been sterilized and enough DEPC-treated, sterile, phosphate buffered saline for the sort (check with us for amount) one day prior to your appointment
Cells can be sorted into a variety of containers. These include 15 ml conical tubes, 12 X 75 mm tubes, or microtiter plates. Test tubes should contain fetal calf serum-300 µl in the 12 X 75 mm tubes and 1 ml in 15 ml conicals. Microtiter wells should contain all the culture medium and feeder cells, if used (usually a volume of 200 µl/well in the 96 well plates).
Remember, if you are not sure about your sample preparation, please call.
For live cells to be cultured after sorting, we recommend that they be processed in our lab. You may leave culture dishes overnight in our CO2 incubator.
All samples to be run on the sorting cytometers must be filtered just prior to sorting or analyzing. We will provide the sterile filters for this purpose.