Step 1. Prepare PBMC, splenocytes, lymph node cells, cell line cells, etc. at a concentration of 2-5 x 107 cells per ml in your favorite FACS buffer (FB, usually PBS + 2% calf serum + 0.1% sodium azide).

Step 2. Add 25ml to each well of a microtiter plate or test tube.

Step 3. Prepare 2x staining cocktail containing all of your labeled reagents at the right titer. The suggested titer for the MHC tetramer reagent is 1:100.

Step 4. Add 25ml of 2x staining cocktail. Mix by pipetting up and down. Avoid bubbles as much as possible, at this and the washing steps.

Step 5. Incubate on ice or other optimized temperature in the dark for 60 minutes.

Step 6. Add 150ml FB to the microtiter wells or 2-3 ml FB if using test tubes. Spin 5 minutes at 1200 RPM. Decant supernatant by "flicking" into sink or bucket of bleach. With infectious/hazardous samples, aspirating may be considered. You may lose more cells if using aspirator.

Step 7. Repeat previous wash step 2 more times.

Step 8. Resuspend cells in 200ml 1% paraformaldehyde (PFA) in PBS and analysis on the flow cytometry.

Tetramer Staining Basic Principles

Principle 1. Keep the staining reactions at as low a volume as possible to conserve reagents. For instance, you can add 25ml of a 2´ stock of stain to 25 ml of a 2´ stock of cells for a total volume of 50ml.

Principle 2. All stainings are carried out at 4°C. Sometimes, higher intensity tetramer stains are obtained if incubated at room temperature or above, but some surface markers — particularly CD62L— are sensitive to the higher temperature. We recommend you investigate staining at 4°C for 30-60 minutes, or room temperature for 30 minutes. Some tetramers have been observed to "fall apart" at 37°C.

Principle 3. Titer your reagents before performing a big experiment. The tetramer stocks are typically used at a final dilution of 1:100, but your mileage may vary.

Principle 4. With some tetramers (but not most), CD8 mediated binding has been observed. In other words, the tetramer binds to all CD8+ cells. The CD8-mediated binding can usually be blocked by inclusion of select CD8 monoclonal antibodies that block the interaction. For instance, Kb tetramers show marked CD8-mediated binding that is blocked by the CT-CD8a antibody from Caltag, leaving intact the specific binding. In other experiments, it seems as if there are steric effects such that as long as the CD8 mAb is labeled with PE (240 kDa), the non-antigen specific effects are abrogated. We are still investigating this.

Principle 5. When CD8-mediated binding is observed, it is necessary to perform a cross-titration experiment with the tetramer and the CD8 antibody to optimize the concentration of each reagent.

Principle 6. For fresh lymphoid cells (PBMC, lymph nodes, splenocytes), we typically stain 1-2x106 cells. For clones and CTL lines, you can probably get away with as few as 2x105 cells. When staining clones, it might be worthwhile to add cells from a clone with a different specificity as an internal negative control.

Please note: Store tetramer product at 4°C. Do not freeze!