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Protein Chemistry Core Lab

Houston, Texas

Protein Core Lab
Protein Chemistry Core Laboratory
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Suggested Staining Protocol

Tetramer Staining Method

  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).
  2. Add 25 µl to each well of a microtiter plate or test tube.
  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.
  4. Add 25 µl of 2x staining cocktail. Mix by pipetting up and down. Avoid bubbles as much as possible, at this and the washing steps.
  5. Incubate on ice or other optimized temperature in the dark for 60 minutes.
  6. Add 150 µl FB to the microtiter wells or 2-3 ml FB if using test tubes. Spin 5 minutes at 1200 RPM. Decant supernant by "flicking" into sink or bucket of bleach. With infectious/hazardous samples, aspirating may be considered. You may lose more cells if using aspirator.
  7. Repeat previous wash step 2 more times.
  8. Resuspend cells in 200 µl 1% paraformaldehyde (PFA) in PBS and analysis on the flow cytometry.

Tetramer Staining Basic Principles

  1. Keep the staining reactions at as low a volume as possible to conserve reagents. For instance, you can add 25 µl of a 2 stock of stain to 25 µl of a 2 stock of cells for a total volume of 50 µl.
  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, room temperature for 30 minutes, and 37 °C for 15 minutes. Some tetramers have been observed to "fall apart" at 37 °C.
  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.
  4. With some tetramers but not most), their binding to the antigen-specific T lymphocytes is influenced by CD8 participation. H-2Kb tetramers have fallen into this category. In some cases, the CD8 mediated non-specific tetramer binding has been observed (the tetramer binds to all CD8+ cells), such as in the presence of 53-6.7 antibody. On contrary, other CD8 monoclonal antibodies could block the tetramer binding, such as the CT-CD8α antibody from Caltag. To eliminate these, we highly recommend that you perform a cross-titration experiment with the tetramer and the desired CD8 antibody to optimize the concentration of each reagent when you use the H-2Kb tetramers.
  5. 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.

 

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