Negative controls allow for precision of immune response monitoring. They assist in defining the specific region or gate for non-specific antigen T cells, enabling accurate measurement of positive (i.e. antigen-specific) T cells.
For example, in a dot plot displaying CD8 (x-axis) and class I tetramer (y-axis), a quadrant gate would be drawn based on the sample stained with a negative tetramer. The upper right quadrant (CD8+Tetramer+) is where the antigen-specific T-cell events will appear when stained with the same panel containing the antigen-specific tetramer.
A suitable negative control for a specific tetramer should consist of three fundamental components:
- Same fluorochrome
- Same MHC allele
- Peptide known to have no reactivity in the sample or in the system studied
For HLA-A*02:01 alleles, MBLI offers a “Negative Tetramer” in BV421 (TB-0029-4), PE (TB-0029-1) or, APC (TB-0029-2). This tetramer is prepared with the HLA-A*02:01 allele and a proprietary peptide whose sequence does not occur in nature. Class I tetramers with “no peptide” cannot be made, as the empty class I molecule is unstable without a peptide.
In the context of the exact flow antibody panel, with the identical staining conditions and acquisition parameters/settings the following options may be considered:
Options Negative controls for human tetramer experiments:
An alternative to match an allele with a specificity known to be unreactive for the donor, or a custom tetramer whose peptide sequence. For Class II alleles, tetramers made using the human CLIP peptide or a non-specific peptide can serve as negative controls.
Another type of negative control is a negative donor.
Options for negative controls for mouse tetramer experiments:
For instance, H-2Kb tetramers that target β-gal or TRP2 have been effectively used as negative controls in experiments involving splenocytes from OT-I mice that express a T cell receptor specifically binding to H-2Kb OVA (SIINFEKL). To ensure accurate gate placement, it is crucial to include the complete panel of antibodies used for the experimental tetramer stain in the control tetramer stain. This accounts for any fluorescent spillover caused by fluorochromes from other channels in the flow cytometer. In experiments with immunised or treated mice, using cells from a naïve mouse stained with the same tetramer/antibody panel as the experimental mouse cells is a suitable negative control.
| Product Code | Product Name |
| TB-0029-1 | iTAg HLA-A*02:01 Negative Tetramer-PE |
| TB-5004-1 | iTAg Tetramer/PE – H-2 Kb TRP2 |
| TS-M008-1 | T-Select H-2Kb Negative Tetramer-SIYRYYGL-PE |
Information provided by MBL.
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