I used to isolate CD8+ cells from PBMC using MACS sorting, then will proceed with FACS sorting of both CD28+CD27+ and CD28-CD27- cells (Double positive and double negative). Before adding the CD8+ microbeads I always use Fc flocking step for 20 min. As I know in-order to avoid the nonspecific florescence in FACS sorting. Here I want to ask weather its mandatory to add Fc blocker? or in case if I don't use blocker do I loose the final cells?
Hi, Fc receptor is widely expressed in B cells, NK cells, macrophages and DCs. So you should add Fc blocker before antibody staining. If you skip this step, you will not lose the last cells, but you will get some unwanted cells that are false positive because of their expression of Fc receptor.
Hello,
If you do not bock Fc Receptors you will most likely have false positive signal and sort other cell types than T cells.
Hello Merlin Jayalal Lawrence Panchali
Using Fc blocker will help. In cell separation, it is important to prevent non-specific binding to ensure good purity of your enriched cells. Fc receptors (FcR) on cells have high affinity for the Fc region of antibodies. To prevent non-specific binding of antibodies to cells, it is advisable to add an FcR blocker to your sample prior to cell separation.
Best.
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