A Peptide-MHC-I Tetramer Staining Technique to Analyze SIV-Specific CD8+ Memory T Cells
Published: January 31, 2024
Abstract
Source: Gonzalez-Nieto, L., et al. Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining. J. Vis. Exp. (2016)
This video demonstrates a method for characterizing CD8+ memory T cells specific to simian immunodeficiency virus (SIV) using peptide-major histocompatibility complex class I (pMHC-I) tetramers. A pMHC-I tetramer is a complex of four MHC-I molecules bound to SIV-specific peptides and conjugated to a fluorophore. These tetramers selectively bind to T cell receptors (TCRs) on SIV-specific CD8+ T cells, facilitating the characterization of these cells.
Protocol
1. PKI Treatment NOTE: This is optional, but recommended for Mamu-B*017:01 tetramers. Resuspend PBMC in R10 medium at a concentration of 1.6 x 107 cells/ml. 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. 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 sus…
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The authors have nothing to disclose.
Materials
| Dasatinib | Axon medchem | Axon 1392 | Must be resuspended in DMSO and immediately stored at -20°C |
| RPMI w/ Glutamax | gibco/ Life Technologies | 61870-036 | Must be stored at 4 °C |
| Heat Inactivated FBS | gibco/ Life Technologies | 10082-147 | Must be stored at 4 °C |
| Penicillin-Streptomycinp-Amphotericin B | Lonza | 17-745E | Must be stored at 4 °C |
| DMSO, Anhydrous | Life Technologies | D12345 | Store at room temperature. |
| 5-mL Round-Bottom Polypropylene Tubes | VWR | 60819-728 | |
| Fluorochrome-conjugated pMHC-I tetramers | NIH Tetramer Core or MBL, Inc. | Must be stored and maintained at 4 °C. Centrifuge at 20,000 x g for 15 min before use. Do not freeze. | |
| Fluorochrome-conjugated mAbs | Various companies | Must be stored and maintained at 4 °C. Do not freeze. | |
| LIVE/DEAD Fixable Aqua Dead Cell Stain Kit | Life Technologies | l34957 | Must be stored at -20 °C. Resuspend each aliquot in 50 μL of DMSO prior to use. |
| Brilliant Stain Buffer | BD Biosciences | 563794 | Must be stored at 4 °C |
| Phosphate Buffered Saline | VWR | 97064-158 | Store at room temperature |
| Albumine Bovine | VWR | 700011-230 | Must be stored at 4 °C |
| Sodium Azide | VWR | 97064-646 | Store at room temperature. Toxic substance. Do not mix with bleach. |
| Bleach | VWR | 89501-620 | Corrosive chemical, cannot be mixed with sodium azide. Handle with care |
| Paraformaldehyde | Electron Microscopy Sciences | 15714-S | Flammable, corrosive, and toxic reagent. Handle with care |
| Polysorbate 20 (Tween-20) | Alfa Aesar | L15029 | Store at room temperature |
| Permeabilization Solution 2 | BD Biosciences | 340973 | Toxic and corrosive reagent. Handle with care |
| Sarsdet Tubes 1.5mL screw top | VWR | 72.692.005 | |
| 2.0ml DNA/RNA Low bind Tubes | Eppendorf | 22431048 | The use of Sterile microtubes is preffered |
| Vortex mixer | |||
| Biosafety Cabinet | |||
| Milli Q Intergral Water Purification system | EMD Millipore | ZRXQ010WW | Molecular Biology grade water from any provider may be used |
| Microcentrifuge | |||
| Centrifuge | |||
| 4 °C refrigerator | |||
| BD LSR II | BD Biosciences | Flow cytometer must contain lasers and filters that are compatible with the staining panel used. | |
| Deionized water | |||
| Aluminum Foil | VWR SCIENTIFIC INC. | 89068-738 | |
| Incubator | Must be able to maintain 37 °C internal temperature | ||
| FACS Diva software | BD Biosciences | ||
| Flowjo software version 9.6 | Flowjo | Used to analyze FCS files generated by FACS Diva software | |
| Micropippette tips |
