Sample Requirements / Sorting / Protocols
General Sample Requirements
TUBES: Cells must be brought in Falcon 12x75mm polystyrene test tubes (Falcon # 2008) for running on any of the analyzers. Other tubes of varying sizes may be used on the cell sorter. Collection tubes for cell sorting should be polypropylene.
VOLUMES and CONCENTRATIONS: Minimum sample volumes per tube are .5 ml. Cell concentrations should be .5 - 1.0 X 106 cells per ml.
FLUOROCHROME SELECTION: Please review the capabilities of the instruments before selecting your fluorochromes and dyes to ensure that the instruments can excite and detect the dyes of choice. For multicolor experiments, it is imperative that you consult fluorochrome guidelines or personnel in the facility for best possible outcomes.
COMPENSATION CONTROLS: Bring positive and negative controls. A negative control is an unstained sample of cells in PBS or fixative or a sample of cells stained only with the secondary ab if this is the system you are using. If you have samples stained with more than one fluorochrome per test tube, bring in a sample stained with each fluorochrome individually. This is for compensation purposes. If you do not have enough positive stained cells for each fluorochrome, you may use antibody capture beads. To make conclusions about your experiment, the proper controls are critical. If you have any questions as to what the appropriate controls are, do not hesitate to call facility personnel at ext. 6908.
General Cell Staining Protocols
Cell Surface Marker/Antigen Staining
For each control and test sample, prepare a single cell suspension of 1X106 cells in cold PBS. Wash cells 1X in at least 1 ml of PBS with 2% FBS/media, centrifuge cells and aspirate supernatant.
Resuspend cells in a final volume of 50ul containing the primary antibody at the correct titer and PBS with 2% FBS/media. Reference manufacturer's guidelines but in general, incubate on ice or at room temp. for 20-30 minutes.
Wash cells 2X in PBS with 2% FBS/media. Centrifuge and aspirate supernatant.
For a directly conjugated antibody go to the next step. For indirect labeling, resuspend cell pellet in a final volume of 50ul containing the Secondary antibody and PBS with 2% FBS/media.
Resuspend cells at a final concentration of 1-2X106 cells/ml in at least 0.4ml fresh 1-2% formaldehyde/PBS. Provide the proper controls and analyze by flow cytometry.
Intracellular Staining- there are also good commercial kits available
- PI Staining of ETOH fixed cells for DNA Content, Cell Cycle and Apoptosis (Easiest Method)
- PI Staining of Nuclei for DNA Content and Cell Cycle (Best Resolution)
There are good commercial kits available. We recommend using kits with Annexin V PE and 7AAD as the best fluorochrome combination.
Current Protocols in Cytometry is available in the Flow Core upon request.
For information on other protocols, visit some of our other links or consult with resources in the Flow Core Facility.