Home › Immunology › Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector
Immunology JoVE (Open Access) Citable · DOI

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

DOI: 10.3791/58680-v
What you'll learn
  • ✓ Perform ex vivo MHC I tetramer staining to identify antigen-specific CD8+ T cells
  • ✓ Collect and prepare single-cell suspensions from blood and lymphoid organs
  • ✓ Analyze cytotoxic T cell responses using flow cytometry after viral vector vaccination
  • ✓ Quantify anti-vector and anti-transgene CD8+ T cell populations simultaneously
Protocol

Here, we present a protocol for the ex vivo qualitative detection of antigen-specific CD8+ T cells. Analysis is possible with single cell suspensions from organs or from small amounts of blood. A broad range of studies require the analysis of cytotoxic T cell responses (vaccination and cancer immunotherapy studies).

Difficulty
advanced
Total time
~4–5 hours per sample batch (collection through flow analysis)
Model organism
Mouse
Biosafety
BSL-2

Steps

1
Collect blood and lymphoid organ tissue

Harvest single-cell suspensions from mouse blood, spleen, or lymph nodes following standard collection protocols. Prepare tissues for downstream staining.

▶ 00:49
2
Prepare staining plate and reagent layout

Organize flow cytometry tubes and staining plates. Establish workflow for antibody and tetramer addition to minimize cross-contamination.

▶ 01:21
3
Prepare MHC I tetramers and fluorescent antibodies

Resuspend MHC I tetramers conjugated to fluorophores and prepare antibody cocktail against CD8 and other markers. Incubate at appropriate concentration and temperature.

▶ 01:59
4
Perform tetramer and antibody staining on samples

Add MHC I tetramer and antibody mixture to cell suspensions. Incubate for specified duration to allow binding of tetramer to antigen-specific TCRs and antibodies to surface markers.

▶ 03:17
5
Lyse red blood cells with hypotonic buffer

Add ammonium chloride lysis buffer to remove erythrocytes while preserving lymphocyte viability. Wash and resuspend cells for flow analysis.

▶ 04:24
6
Acquire and analyze flow cytometry data

Run stained samples on flow cytometer. Gate on live CD8+ T cells and identify antigen-specific populations via tetramer-positive events. Quantify frequencies of anti-vector and anti-transgene CD8+ subsets.

▶ 05:09
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