A Peptide-MHC-I Tetramer Staining Technique to Analyze SIV-Specific CD8+ Memory T Cells

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.

1. Resuspend PBMC in R10 medium at a concentration of 1.6 x 10⁷ cells/ml.
2. 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.
3. 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 suspension containing 8.0 x 10⁵ PBMC and 50 nM of PKI.
4. Proceed to the pMHC-I tetramer staining step.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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