Practical advice to set up and troubleshoot your flow cytometry experiment for immune monitoring.
Unexpected Cell Populations are Observed
It is possible to observe unwanted cell populations when using Dextramer® reagents to detect antigen-specific cells. A Dextramer® reagent carrying a peptide-MHC I complex should theoretically only interact with CD8+ T cells, however, the reagent may associate with other cells. The biology of this interaction is not well understood.
The gating strategy can be adjusted to focus analyses on the desired cell population. Include a DUMP channel as part of your gating strategy to clean up the dataset. Some typical DUMP markers in Dextramer® assays are CD56/CD16 for NK cell and granulocyte removal, CD14 for monocyte removal, and CD19 for B cell removal. Make sure to also remove dead cells from the analysis. Thus, the gating strategy for a clearly resolved antigen-specific CD8+ T cell population may be:
- steady flow
- lymphocyte selection by scattering pattern, excluding debris for the analysis
- single cells
- live/CD3+ cells
- DUMP+/CD3+
- CD8 vs. Dextramer®
Remember that gating is an iterative process. Back gating can be useful to confirm that the final population is legitimate or to further optimize set gates.
Further Help with Troubleshooting Flow Cytometry
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MHC Dextramer® Reagents
Sensitively quantify target immune cells by flow cytometry with a strong signal-to-noise ratio.