Procedures and Protocols
For flow cytometry:
Samples used on the facility cytometers must be clump-free. Passing cells through a 40 or 70 µm mesh should clean most samples. A good analysis requires that users bring the appropriate controls: a negative, unstained sample (if secondary antibodies are used, samples with the secondary antibody only should also be brought) and single-stained samples for each antibody used in the experiment. Samples should have at least 50 000 cells and be resuspended in at least 300 µl of PBS (500 µl for the LSRFortessa).
For cell sorting:
Cells should be resuspended in sterile PBS containing less than 2% serum. As with all flow cytometry experiments, controls are essentials. Users are required to bring an unstained sample and single-stained controls for each of the antibody used or the sorting will not be done. A single-cell suspension is absolutely vital and cells should be passed through a 30-50 µm cell strainer before coming to the facility. Cells suspensions containing aggregates will not flow evenly through the sorter and will likely block its nozzle, causing the sorting to stop. This will lengthen the sorting time and might results in the loss of some of the sample. Cells can be sorted in Eppendorfs, 5 or 15ml tubes and collected in plates (6 to 96-well), microscope slides or 5 or 15ml polypropylene tubes. The use of polystyrene tubes is not recommended for sample collection.
Not sure about which fluorochrome combination to use? Here's a list of frequently used fluorochromes and their respective detectors on our flow cytometers Fluorescence Parameters
FACSCalibur SOP facscalibur_sop.pdf
FACSCanto II SOP facscanto_ii_sop.pdf
LSRFortessa SOP lsr_fortessa_sop.pdf