Sample Preparation:
Requirements of sample preparation:
Samples should be prepared in a single-cell suspension at a concentration of 4
~25 million cell/ml, however. preservative such a sodium azide should NOT be used. Cells must be filtered through 35-70 um filters just prior to sorting in order to remove any clumps or aggregates. Check websites for BD Biosciences and GCAT to get order information for filters.
Prepare your cells as you normally would for flow cytometry analysis. Cells can be suspended in PBS or serum-free culture media (no phenol red added) with a small amount of BSA or FBS (0.5% or 2%) and 2mM EDTA to keep cells viable and non-aggregated.
Please do test experiments on flow cytometry to to confirm staining an percentage of your target cells.
The facility cannot accept potentially pathogenic samples for cell sorting. I you have questions regarding viable human/primate samples or lentivirus-treated samples, please consult with the core members. Samples labeled with radioactive material are not accepted under any circumstances.
What to bring:
Cells must be kept on ice and 5 ml polypropylene round-bottom tubes 12x75mm style (Falcon #352063 from VWR).
Negative control (no staining cells, 0.5 million/0.5 ml).
Single staining cells of each fluorochrome to set compensation (if multiple staining applied, 0.5 million cells/0.5 ml).
Cells to be sorted (4-25 million cells/ml).
Additional 5 ml polypropylene round bottom tubes and cell strainer/filters.
Cell suspension medium/buffer.
Several 5ml round bottom tubes filled with 1ml of culture medium or 50 ml tubes filled with 10 ml of culture medium to collect sorted cells.
A print out of the data obtained on the flow cytometry analyzer (FC 500) which shows your population(s) of interest.
Sorting Rates:
Suggested sorting rate for cells that are large, fragile or easily aggregate is < 6000/second using 100 micron nozzle.
Smaller cells can be sorted at 12,000
~15,000/second, using 70 micron nozzle.