Importance of Using an FcR Blocker During Cell Separation Procedures
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. For human and mouse samples, our EasySep™ cell separation kits contain an appropriate FcR blocker. Unfortunately, there isn't a universal blocker which will work for all species, so if you are working with a species other than human or mouse, you will have to add a blocker appropriate to the species with which you are working.
For best results, we recommend using one of the following as an FcR blocker during cell separation procedures:
- Use normal serum from the species in which your primary antibody was raised. For example, if you are using a mouse anti-rhesus CD-x, then you should use normal mouse serum, or if you are using a donkey anti-rhesus CD-x, then you should use normal donkey serum. Typically, 5% serum should be sufficient for blocking.
- Use an anti-CD32 (FcgRII) or anti CD16/32 (FcgR III/II) antibody clone which cross-reacts with your species of interest. For example, if you are working with rhesus samples, Anti-Human CD32 Antibody, Clone FLI8.26 (Catalog #60135) is appropriate. However, please use caution if using these antibodies as the FcR blocker, and make sure that this antigen is not expressed on your cell type of interest. If you are isolating monocytes, macrophages, granulocytes, or some lymphocytes, using serum may be a better option, otherwise the selecting antibody's target (i.e. CD32) will be blocked and selection will fail.
The EasySep™ Other Species indirect selection kits can be used to isolate cells of interest from any species. Simply label the desired cells with an appropriate primary antibody which is conjugated to biotin, PE, FITC or APC. Then you can use the corresponding EasySep™ kit (see kits listed below), along with the appropriate FcR blocker to ensure good purity of your isolated cell population.