Question: How do I pre-screen antigens or peptides to check their quality for Barcode Enabled Antigen Mapping (BEAM)?
Answer: A protocol for pre-screening antigens or peptides is available in the BEAM Reagent Assembly, Sample Labeling and Flow Sorting User Guide (CG000595). The goal of pre-screening is to identify low quality antigens/peptides, such as those with low purity, low solubility, improper biotinylation (BEAM-Ab) or improper loading into the MHC (BEAM-T).
We recommend performing pre-screening to test every antigen or peptide prior to performing the actual BEAM-Ab or BEAM-T experiment. Each antigen or peptide only needs to be screened once. Antigens/peptides that do not pass pre-screening should not be used in the actual BEAM experiment.
In the pre-screening protocol, cell samples are labeled separately with individual BEAM Assemblies prepared from each antigen or peptide. After labeling, each cell sample is analyzed by flow cytometry. Poor quality antigens/peptides can lead to a shift in PE signal, with many cells appearing as PE-positive. Examples of flow cytometry plots from pre-screening experiments are shown in the Appendix of the BEAM Reagent Assembly, Sample Labeling and Flow Sorting User Guide.
The pre-screening protocol can be performed using a non-experimental cell sample. Identifying low quality antigens/peptides does not require antigen-specific cells to be present. Alternatively, it is possible (but not required) to use an experimental sample for pre-screening. This would allow you to assess reactivity to the antigen/peptide of interest. However, it is not a requirement to run an experimental sample through the pre-screening workflow. Alternative methods, such as ELISpot, can be used to confirm that your experimental samples have reactivity to your antigens or peptides of interest.
For BEAM-Ab, see additional guidance here: How can I reduce background PE signal for the BEAM-Ab Negative Control Assembly?
Products: Single Cell Immune Profiling, Barcode Enabled Antigen Mapping (BEAM)