MHC Tetramer staining method

Human whole blood samples

• Collect blood by venipuncture into a blood collection tube containing an appropriate anti-coagulant.
• Add 10 µL of T-Select MHC Tetramer to each 12 x 75 mm test tube.
• Add 200 µL of whole blood into each test tube.
• Vortex gently.
• Incubate for 30-60 minutes at 2-8°C or room temperature (15-25°C) protected from light.
• Add any additional antibodies (e.g. anti-CD8) and vortex gently.
• Incubate for 30 minutes at 2-8°C protected from light.
• Lyse red blood cells using commercially available reagents.
• Prepare samples according to description of the package insert.
• Store prepared samples at 2-8°C protected from light for a minimum of 1 hour (maximum 24 hours) prior to analysis by flow cytometry.

Human Peripheral Blood Mononuclear Cells (PBMC) samples

• Prepare peripheral blood mononuclear cells (PBMC) according to established procedures. Cells should be re-suspended at a concentration of 2 x 10⁷ cells/mL. 50 µL of sample is required for each T-Select MHC Tetramer determination.
• Add 10 µL of Clear Back (human FcR blocking reagent, Code No. MTG-001) to each 12 x 75 mm test tube.
• Add 50 µL PBMC into each test tube (e.g. 1 x 10⁶ cells per tube).
• Incubate for 5 minutes at room temperature.
• Add 10 µL of T-Select MHC Tetramer and vortex gently.
• Incubate for 30-60 minutes at 2-8°C or room temperature (15-25°C) protected from light.
• Add any additional antibodies (e.g. anti-CD8) and vortex gently.
• Incubate for 30 minutes at 2-8°C protected from light.
• Add 3 mL of PBS or FCM buffer (2% FCS/0.09% NaN₃/PBS).
• Centrifuge tubes at 400 x g for 5 minutes.
• Aspirate or decant the supernatant.
• Resuspend the pellet in 500 µL of PBS with 0.5% formaldehyde.
• Store prepared samples at 2-8°C protected from light for a minimum of 1 hour (maximum 24 hours) prior to analysis by flow cytometry.

Mouse spleen samples

• Collect lymph node, spleen or thymus and prepare a single-cell suspension according to an established protocol. Cells should be re-suspended at a concentration of 2 x 10⁷ cells/mL. 50 µL of sample is required for each T-Select MHC Tetramer determination.
• To each 12 x 75 mm test tube add 10 µL of Clear Back (human FcR blocking reagent, Code No. MTG-001).
• Add 50 µL cell suspension into each test tube (e.g. 1 x 10⁶ cells per tube).
• Incubate for 5 minutes at room temperature.
• Add 10 µL of T-Select MHC Tetramer and vortex gently.
• Incubate for 30-60 minutes at 2-8°C protected from light.
• Add any additional antibodies (e.g. anti-CD8) and vortex gently.
• Incubate for 30 minutes at 2-8°C protected from light.
  If red blood cell lysis is necessary, proceed to step 8-16 in the Procedure for Whole Blood section. If red blood cell lysis is not necessary, continue to step 9 below.
• Add 3 mL of PBS or FCM buffer (2% FCS/0.09% NaN₃/PBS).
• Centrifuge tubes at 400 x g for 5 minutes.
• Aspirate or decant the supernatant.
• Resuspend the pellet in 500 µL of PBS with 0.5% paraformaldehyde or formalin.
• Store at 4°C protected from light for a minimum of 1 hour (maximum 24 hours) prior to analysis by flow cytometry.

Staining differences among CD8 antibody clones

Human CD8 antibody clones

Mouse CD8 antibody clones

Splenocytes from mice transgenic for OVA-specific T cells (OT-I) were used to explore staining differences among mouse CD8 antibody clones in combination with H-2K^b Tetramers. OT-I mouse spleen cells (1 × 10⁶ cells/sample) were stained with H-2K^b OVA Tetramer-PE (10 µL/sample) and serially diluted anti-mouse CD8 (clone KT15 or clone 53.6.7) antibody in a final assay volume of 100 µL. H-2K^b β-galactosidase (β-gal) Tetramer-PE (10 µL/sample) was used as a negative tetramer to assess non-specific binding. When anti-CD8 clone 53.6.7 was used, positive staining was observed on CD8 positive cells with both OVA Tetramer and β-gal Tetramer. Even when 53.6.7 was diluted, the cells were stained with both tetramers similarly, suggesting the tetramer staining was not specific.
When anti-CD8 clone KT15 was used, however, CD8 positive cells among OT-I mouse spleen cells were specifically stained only with OVA Tetramer at all antibody concentrations tested. Cells stained with β-gal Tetramer were negative. Titration of KT15 revealed an optimal signal to noise ratio at 0.63 µL/sample for OT-I splenocytes. In summary, when staining cells with H-2K^b tetramers it is important to use appropriately titrated anti-CD8 clone KT15 and include a negative control tetramer in the experiment.

Mouse spleen cells prepared using peptide immunization for OVA-specific CTL induction were stained with H-2K^b OVA Tetramer-PE (10 µL/sample) and anti-mouse CD8 (clone KT15 or clone 53.6.7) H-2K^b β-galactosidase Tetramer-PE (10 µL/sample) was used as a negative tetramer to assess non-specific. When KT15 was used, OVA-specific tetramer positive cells were observed compared with the negative tetramer, while when 53.6.7 was used, marked non-specific staining was observed in both H-2K^b β-galactosidase Tetramer samples clone 53.6.7 is known to have poor compatibility with OVA and other H-2K^b Tetramers. MBL recommends the use of KT15 for murine CD8 staining.