A Peptide-MHC-I Tetramer Staining Technique to Analyze SIV-Specific CD8⁺ Memory T Cells

1. PKI Treatment

NOTE: This is optional, but recommended for Mamu-B*017:01 tetramers.

1. Resuspend PBMC in R10 medium at a concentration of 1.6 x 10⁷ cells/ml.
2. Add 50 µl of this cell suspension to the corresponding flow cytometry tubes. Add 50 µl of a 100 nM solution of PKI to each tube.
3. Vortex each tube. Incubate at 37 °C for 30 min. By the end of this step, each tube should have 100 µl of a cell suspension containing 8.0 x 10⁵ PBMC and 50 nM of PKI.
4. Proceed to the pMHC-I tetramer staining step.

2. Staining with Fluorochrome-labeled pMHC-I Tetramers

1. Before preparing the pMHC-I tetramer master mix, centrifuge the pMHC-I tetramer tubes at 20,000 x g for at least 15 min at 4 °C. The goal of this step is to pellet protein aggregates in the pMHC-I tetramer solution that may increase background staining. Once the centrifugation step is done, avoid pipetting from the bottom of the tube where the protein aggregates will have accumulated.
2. Prepare enough pMHC-I tetramer master mix to stain all experimental tubes. Prepare an excess of 15% of the total volume of this master mix to account for pipetting errors.
3. Dilute pMHC-I tetramers in stain buffer (e.g., Brilliant Stain Buffer) so that 25 µl of the pMHC-I tetramer master mix is added to each test.
   1. Label PBMC with two pMHC-I tetramers per test; one conjugated to allophycocyanin (APC) and the other to Brilliant Violet (BV) 421.
4. Add 8.0 x 10⁵ PBMC to the corresponding flow cytometry tubes in a final volume of 100 µl. If the cells were subjected to the PKI treatment described above, they should already be resuspended in 100 µl at this step.
5. Add 25 µl of pMHC-I tetramer master mix to the corresponding flow cytometry tubes. By the end of this step, the final volume in each tube should be 125 µl.
6. Vortex each tube in order to homogenize the cell suspension.
7. Incubate in the dark at room temp for 45 min. Prepare the surface-staining monoclonal antibody (mAb) cocktail during this 45-minute incubation.

3. Surface Staining

1. Prepare enough mAb master mix to stain all experimental tubes. Prepare an excess of 15% of the total volume of this master mix to account for pipetting errors.
2. Adjust the volume of the master mix with stain buffer so that 50 µl is added per test.
3. For proper exclusion of non-CD8⁺ T-cells and delineation of memory subsets, use titrated amounts of mAbs directed against the following molecules in the surface staining master mix.
   NOTE: The fluorochromes conjugated to each mAbs are provided as a reference: CD14 BV 510, CD16 BV 510, CD20 BV 510, CD8α BV 785, CD28 PE Cy7, and CCR7 FITC. Note that the mAbs against CD14, CD16, and CD20 are conjugated to the same fluorochrome (i.e., BV 510) since they will be included in the "dump" gate.
4. Include a fixable dye for discriminating dead cells in the surface staining master mix [i.e., amine-reactive dye (ARD)]. Make sure that the ARD reagent is conjugated to a fluorochrome with a similar emission spectrum as the ones used in the "dump" gate. In this case, use ARD Aqua.
5. Add 50 µl of the staining master mix described in 3.1-3.4 to the corresponding flow cytometry tubes. By the end of this step, the final volume in each tube should be 175 µl.
6. Vortex each tube. Incubate in the dark at room temp for 25 min.
7. Wash cells with wash buffer (phosphate-buffered saline (PBS) solution containing 0.1% of bovine serum albumin and 0.45 g/L NaN3).
   CAUTION: Sodium azide (NaN₃) is a toxic substance. Exposure to even minute amounts can cause symptoms. Handle this substance according to the guidelines specified by the Environmental Health and Safety (EHS) Office at the institution where these experiments are being performed.
8. Centrifuge tubes at 510 x g for 5 min. Carefully decant supernatant into a separate waste container. Be sure not to disturb the pellet.
   CAUTION: Do not decant wash buffer supernatant into reservoirs containing bleach as it can react with the sodium azide present in the wash buffer and result in the formation of a toxic gas. Contact the EHS Office at the institution where these experiments are being performed for guidelines on how to dispose of sodium azide.
9. After decanting, vortex the cells in the leftover liquid retained in each tube.

4. Cell Fixation

1. Add 250 µl of a 2% paraformaldehyde (PFA) solution to all tubes to fix the cells.
   CAUTION: PFA is a toxic substance. Exposure to even minute amounts can cause symptoms. Handle this substance according to the guidelines specified by the EHS Office at the institution where these experiments are being performed.
   1. Since the 2% PFA solution must be isotonic to the cells, use PBS to prepare this solution. One hundred milliliters of a 2% PFA solution is enough for multiple experiments. Thoroughly vortex all tubes immediately after the addition of 2% PFA in order to prevent the formation of cell aggregates.
2. Incubate in the dark at 4 °C for 20 min.
3. Wash cells by repeating steps 3.7-3.9. Once this step is completed, the cells can be stored in the dark at 4 °C for 24-48 hr. Before proceeding to the Permeabilization phase, vortex the tubes thoroughly.

5. Permeabilization of Cells

1. Add 500 µl of permeabilization buffer. Vortex all tubes.
2. Incubate in the dark at room temp for 10 min.
3. Wash cells by repeating steps 3.7-3.9.

6. Intracellular Staining

1. Prepare enough mAb master mix to stain all experimental tubes.
2. Adjust the volume with PBS or stain buffer so that 50 µl of the intracellular mAb master mix is added per test.
3. Prepare the mAb master mix described in 6.1 and 6.2 using titrated amounts of mAbs directed against CD3 and granzyme B (Gzm B).
   NOTE: TCR engagement by pMHC-I tetramers can result in CD3 internalization, which can interfere with the detection of tetramer + CD3⁺ CD8⁺ T-cells if the anti-CD3 mAb is added to the surface staining master mix. To avoid this, add the anti-CD3 mAb at this stage, that is after the cells are permeabilized. The fluorochromes conjugated to each mAb are listed as a reference: CD3 PerCP Cy5.5 and Gzm B PE.
4. Add 50 µl of mAb cocktail to the corresponding tubes. Incubate in the dark at room temp for 30 min.
5. Wash cells by repeating steps 3.7-3.9. The tubes are ready to be acquired in a flow cytometer.