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…

開示

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

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記事を引用
A Peptide-MHC-I Tetramer Staining Technique to Analyze SIV-Specific CD8+ Memory T Cells. J. Vis. Exp. (Pending Publication), e21921, doi: (2024).

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