Question: Can I use two fluorophores to identify antigen-positive cells in a Barcode Enabled Antigen Mapping (BEAM) experiment?
Answer: In the BEAM assay, antigen-positive cells are identified using the PE fluorophore on the 10x Genomics BEAM Conjugate. It may be possible to label cells with antigens conjugated to a second fluorophore to increase the specificity of flow sorting. We have performed limited testing using Biolegend Streptavidin APC (catalog number 405207)* as a second fluorophore, as outlined below. This is not officially supported and may require additional optimization.
The following protocol modifications are suggested for dual labeling of cells with an antigen-Streptavidin-APC complex in addition to the BEAM Assembly (PE).
- Conjugate the biotinylated antigen to Biolegend Streptavidin APC by following the volumes outlined in CG000595, but using 2.5-3.5 ul Biolegend Streptavidin APC (405207) instead of 2.0 ul BEAM Conjugate.
- Label cells simultaneously with equal volumes of the BEAM-Ab Assembly (PE) and the antigen-Streptavidin-APC complex.
- Generate peptide-MHC multimers with Biolegend Streptavidin APC by following the volumes outlined in CG000595, but using 2.5-3.5 ul Biolegend Streptavidin APC (405207) instead of 2.4 ul BEAM Conjugate.
- Label cells simultaneously with equal volumes of the BEAM-T Assembly (PE) and the peptide-MHC-Streptavidin-APC complex.
In the figure below, cells were labeled with BEAM-Ab Assemblies, a B cell flow antibody panel, and 7AAD, as outlined in CG000595. Flow gates were set to sort single cells > live cells (7-AAD negative) > lineage negative > CD19+ > PE+ cells. In the left panel, cells labeled with the BEAM-Ab Assembly can be identified as PE positive. In the right panel, cells were also labeled with Streptavidin-APC, and dual positive cells (PE+, APC+) can be identified. These results suggest that Streptavidin-APC is compatible with the BEAM assay.
In our limited testing, adding Streptavidin-APC did not lead to any major differences in overall BEAM assay performance. However, results may vary based on the sample type and frequency of antigen-positive cells.
* Additional considerations regarding Biolegend Streptavidin APC:
- The volume recommendations in this article apply to Biolegend Streptavidin APC, catalog number 405207, which is provided at a concentration of 0.2 mg/ml. The volumes will need to be adjusted accordingly if a reagent with is higher concentration is used (eg. Biolegend APC Streptavidin High Concentration, catalog number 405243, which is provided at a concentration of 1 mg/ml).
- Non-barcoded Biolegend Streptavidin APC was tested. This may be used to increase the specificity of flow sorting, but does not contain a Feature Barcode oligonucleotide and therefore will not be incorporated into the BEAM library.
Products: Single Cell Immune Profiling, Barcode Enabled Antigen Mapping (BEAM)