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 7K/sec.
You will need to bring cell media in the tubes or trays into which you want to sort cells. Tubes should be polypropylene and can vary in size. Media should fill about 1/3 of the tube space before collection begins.
It is also highly recommended that you filter your cells before sorting if your cells are clumping together. Clogs will be a problem to any sorting experiment. 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.
Special Requirement for Sorting Primary Human Cells:
For sorting live, primary human cells in the Flow Cytometry Core, it is required that the patient/normal donor be tested for HIV Ab and Hep. C and B Core Ab. (This requirement does not necessarily apply to human cells that are from an established published cell line.)
This process will include obtaining the appropriate consent from the patient/normal donor. Normal donors who are employees of HMC may arrange to have this testing done through Employee Health. For more information on this process regarding patient studies, you may contact:
Terry Novchich, Clinical Trials Coordinator
Primary Phone: 717.531.1389
Alternate Phone: 717.531.3779
Mail Code: H052
Building: Main Building
Room Number: MB C662B
As of 2008, these are the quoted prices from Clinical Labs for these tests required are as follows:
For NIH funded studies: Hep B Core Ab $21.88
HIV Ab $22.93
Hep C $27.68
For all other studies: Hep B Core Ab $57.12
HIV Ab $33.95
Hep C $40.35
These prices are subject to change, depending on the Finance costing procedure and changes to the NIH contract.
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. This requirement is in addition to the Cell Sorting Experiment Form required for all cell sorting tests.