Question: Should I include negative controls in the Barcode Enabled Antigen Mapping (BEAM) assay?
Answer: Yes. A Negative Control Assembly should be included in every BEAM experiment. Negative controls are required for Antigen Specificity scoring in Cell Ranger. The Antigen Specificity score is calculated by comparing the difference in signal between the negative control antigen and the antigen of interest.
- In the BEAM-Ab assay, the Negative Control Assembly is generated without any antigens (using PBS and the BEAM Conjugate alone) or using an off-target antigen (mouse or human serum albumin). This will allow you to identify cells that bind non-specifically to streptavidin or PE.
- In the BEAM-T assay, the Negative Control Assembly is generated using an MHC-matched Negative Control peptide that is provided with each 10x Genomics MHC Monomer Kit. This will allow you to identify cells that bind non-specifically to streptavidin, PE or the MHC Monomer.
- The Negative Control peptides are non-targeting peptides with a proprietary sequence. Each Negative Control peptide was optimized for the specific MHC allele.
- We do not recommend using Assemblies prepared from empty/unloaded MHC monomers (without a peptide) as a negative control. Unloaded Assemblies can lead to a shift in PE signal, with many cells appearing to be PE positive.
- An Unloaded Assembly should be used during the BEAM-T pre-screening protocol only. It should not be used in the actual BEAM-T experiment. Refer to the Appendix of CG000595 for an example of a flow cytometry plot from an Unloaded Assembly in the BEAM-T pre-screening experiment.
Products: Single Cell Immune Profiling, Barcode Enabled Antigen Mapping (BEAM)