Tetramer Staining Basic Principles
- 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.
- 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.
- Titer your reagents before performing a big experiment. The tetramer stocks are typically used at a final dilution of 1:100 to 1:200, but your mileage may vary.
- 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.
- 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.
- 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. You can do this at a ratio of 10:90 specific clones: non-specific clone.