Although MHC tetramer staining of antigen-specific T cells for flow cytometry is widely recognised for its high specificity, it is important to acknowledge the potential for non-specific staining. To address this challenge, investigators have developed a gating strategy, illustrated below, that effectively minimises non-specific staining by incorporating a dump channel. The dump channel is tailored to each experiment and consists of a pool of antibodies against cells that do not express our specific targets. For instance, CD8+ T cells can be eliminated from our targeted gate by using anti-CD4/anti-CD14/anti-CD19 antibodies, all of which can be labelled with the same fluorochrome.
Excellent Staining with Minimal Background
First, we gate the lymphocytes using forward and side scatter (upper left). Then, we gate the single cells using forward scatter height and wide, followed by side scatter height and wide (upper middle and right). Live cells are then gated using a forward scatter area and 7-AAD (live dye) negative cells (middle left). Next, we gate CD8+ cells by excluding the dump channel (CD4, CD14, and CD19) negative cells and selecting CD3+ cells (middle right). To determine the CMV tetramer positive gate, we use non-peptide-stimulated PBMCs. The gate is set on CD8+ cells that are negative in the tetramer channel (lower left). This gate is used to identify CD8+ tetramer+ cells in a CMV peptide-stimulated sample (lower right).
| Product Code | Product Name |
| TB-CMVmv-1 | Multi-Allele CMV Tetramer-PE (7 vials) |
| TB-0029-1 | iTAg HLA-A*02:01 Negative Tetramer-PE |
Information provided by MBL.
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