Question: Which antibodies should I include in my fluorescent antibody panel for flow sorting in the Barcode Enabled Antigen Mapping (BEAM) assay?
Answer: In the BEAM assay, cells should be labelled with a fluorescent antibody panel that includes the following antibodies:
- Antibodies to select for the cell type of interest:
- CD8 and CD3 antibodies are used to select for cytotoxic T cells in the BEAM-T assay.
- CD19 antibody is used to select for B cells in the BEAM-Ab assay.
- Antibodies to select against other lineages:
- These antibodies are used to exclude other cell types, such as Natural Killer cells, erythroid cells, monocytes, dendritic cells. These antibodies are used for the "lineage negative" or "dump" channel during sorting.
- Including these antibodies helps to increase the specificity of the sort by eliminating cells from other lineages that may non-specifically bind to the BEAM Assemblies.
Cells should also be labeled with a live dead marker. In-house, we have tested 7-AAD. Using other live dead markers may be possible, but we have not tested this in the BEAM assay.
Recommended vendors and part numbers for the antibodies and 7-AAD can be found in the BEAM Protocol Planner (CG000590). Additional guidance can also be found in the BEAM Flow Cytometry Guidelines Technical Note (CG000598). Example antibody panel for BEAM-T and BEAM-Ab, mouse and human, are shown here:
Products: Single Cell Immune Profiling, Barcode Enabled Antigen Mapping (BEAM)