5/29/2023 0 Comments Purity definition![]() In order to obtain an accurate assessment of purity, it is important to gate out cellular debris and dead cells during analysis of the sample. If samples cannot be analyzed immediately, fix with 1% paraformaldehyde and store for up to two weeks at 2 – 8® protected from light. Pour off supernatant and resuspend the pellet in 100-500 µL of PBS or FACS sheath fluid. Incubate at 2 – 8®C or on ice for 30 minutes in the dark.This step allows dead cells to be gated out for more accurate flow cytometry analysis. If desired, add a viability stain such as propidium iodide (PI) or 7AAD to each sample.To the second tube, add the appropriate fluorescentlyconjugated isotype control antibody (e.g.The volume will typically be 5-20 µL of antibody per test. To the first tube, add the appropriate fluorescently-conjugated monoclonal antibody or antibodies (see Table 1) according to the antibody manufacturer’s instructions.After cell separation, place 100 µL of enriched cells into two separate 5 mL FACS tubes (cells should be at a concentration between 1 x 10 6 and 1 x 10 7 cells/mL).Step-by-Step Protocol: Purity Assessment by Flow Cytometry Consult the appropriate PIS for detailed instructions. Staining procedures for purity assessment are provided in the Product Information Sheets (PIS) for all EasySep™ and RoboSep™ isolation kits (see product list on final page). Samples separated by this method can be stained using antibodies against the primary cell surface marker (e.g. Negative selection isolates unlabeled target cells. These antibodies will bind to the antibodies used for positive selection. Use a fluorochrome-conjugated secondary antibody, such as a FITC-labeled goat anti-mouse IgG.PE-labeled) targeting the primary cell surface marker at the same time as the selection cocktail. Add a fluorochrome-conjugated antibody (e.g. ![]()
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