Cell Sorting Requirements and Tips
Cell Sorting Requirements
Cells should be concentrated to at least 5 million cells/ml. If you want to sort lots of cells in a short amount of time, you may concentrate your cells even more (but the more concentrated your cells are, the more likely they might form clumps). Remember to calculate how many total cells you will need to analyze in order to sort the required number of target cells you want to have after the sort experiment. In general, lymphocyte sized cells can be run at rates close to 30K/sec. and cell lines can be run around 12K/sec.
You will need to bring cell media in the tubes or trays into which you want to sort cells. Tubes should be polypropylene to avoid static charges and can vary in size. Media should fill about 1/3 of the tube space before collection begins.
It is required that you filter your cells before sorting. Clogs will be a problem to any sorting experiment and increase biohazard risks. 35 or 40um filter cap tubes are available from BD Biosciences for this purpose.
Also, you must read and adhere to the SAFETY GUIDELINES before sorting unfixed cells and you must fill out and have the P.I. sign the CELL SORTING EXPERIMENT form before you can schedule any sorting experiment. This form must be given to the core personnel for review ONE WEEK prior to scheduling your sorting experiment.
If, after our review of the CELL SORTING EXPERIMENT form, it is determined that your experiment is at the BSL2+ level, you will be required to read and adhere to the Flow Cytometry BLS2+ SOP for cell sorting which is available in the cell sorter room.
Special Requirement for Sorting Primary Human Cells
Any request to sort live, primary human cells in the Flow Cytometry Core will require a review of the samples in question by the Flow Core Director based on the information on the CELL SORTING EXPERIMENT form and possible consultation with the PI involved. (This does not necessarily apply to human cells that are from an established published cell line.)
Several outcomes of this evaluation are possible. Some high risk populations may be deemed too high-risk to sort in our facility. Other populations may be sorted under BSL2 conditions, others under BSL2+ conditions and still others only after the donors are tested for HIV antibody and Hepatitis C and B Core antibody.
If donor testing is required, you must contact the Clinical Trials Coordinator for more information on this process. Once the tests are completed, a copy of the test report without patient ID will be signed by the requesting PI and forwarded to the Flow Cytometry Facility before the cell sorting experiment is performed.