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      Tetrameric MHC staining protocol for flow cytometry

      Application: MHC Tetramers enable direct detection and specific isolation of antigen-specific T cells by FACS. Kactus proprietary prMHC (peptide-ready MHC) tetramers can be loaded with any target neoantigen in a simple loading protocol. The peptide loading is highly efficient. The loaded prMHC tetramer, which contains fluorescent labeling can be used immediately without dialysis for cell sorting.

      Materials and Instruments

      • Instruments

      NovoCyte Flow Cytometer (Agilent, NovoCyte 2000R) is the one we used in-house. Any FACS machine is okay as long as they have proper channels, like APC, FITC, or PE.

      • Reagents
      1. APC-equivalent Human Peptide Ready HLA-A*02:01&B2M Tetramer Protein (MHC-HM43RTC)
      2. Dasatinib (MedChemExpress, Catalog Number: HY-10181) We dissolve Dasatinib with DMSO to the concentration of 100mM. You may need to dilute several times because the final working concentration is 50nM in this protocol.
      3. FACS Buffer eBioscience™ Flow Cytometry Staining Buffer (thermofisher, 00-4222-26) or equivalent. To make in-house FACS Buffer, PBS + 2%FBS (Biological Industries, 04-001-1ACS) is the recipe for FACS buffer. Any FBS is okay.
      4. Cell lines expressing TCRs. Can be natural/engineered T cells or cells expressing exogenous TCRs.

      Experimental Procedure

      1. Transfer 1x106 TCR-expressing cells into 1.5ml tube for each sample.
      2. Cells should be resuspended in 100 µL FACS Buffer containing a suitable Fc receptor block, such as FBS.
        • Centrifuge cells at 1000 rpm for 2 minutes. Discard the supernatant.
        • Resuspend cells with 200-400 µL FACS Buffer. Then centrifuge cells at 1000 rpm for 2 minutes. Discard the supernatant. Repeat this step 2 times.
        • Resuspend Cells using 100 µL FACS Buffer.
      3. Add Dasatinib (final concentration in the system: 50nM) into cells and incubate on ice for 30 minutes.
      4. Centrifuge tubes at 1000 rpm for 2 minutes and resuspend cells with 200 µL FACS Buffer.
      5. Centrifuge tubes at 1000 rpm for 2 minutes and resuspend cells with 100 µL FACS Buffer.
      6. Add APC-equivalent MHC tetramers (Recommended final concentration: 20-60 µg/mL) into the tube.
      7. Incubate the mixture on ice for 1 hour.
      8. Add 200-400 µL FACS Buffer into the cell-tetramer mixture. Centrifuge at 1000 rpm for 2 minutes. Discard the supernatant. Repeat this step 2 times.
      9. Resuspend cells with 200-300 µL FACS Buffer.
      10. Analyze cell population by flow cytometry.