Selective Isolation of CD4+ Vδ1+ γδ T Cells from Human Peripheral Blood Mononuclear Cells

Abstract

Source: Welker, C., et al. Isolation and Ex Vivo Culture of Vδ1⁺CD4⁺γδ T Cells, an Extrathymic αβT-cell Progenitor. J. Vis. Exp. (2015).

This video demonstrates a protocol for the magnetic labeling-based isolation of a low-concentration population of CD4⁺Vδ1⁺γδ T cells from human peripheral blood mononuclear cells (PBMCs). Using a combination of FITC-conjugated anti-Vδ1 antibodies, magnetic anti-FITC microbeads, and anti-CD4 antibody-coated superparamagnetic beads, the CD4⁺Vδ1⁺ T-cells can be successfully purified from the PBMC population.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Isolation of Vδ1 T Cells

1. Take <1 x 10⁶ isolated lymphocytes for an initial FACS determining the frequency of Vδ1 CD4 T cells.
   NOTE: The population size of this T-cell entity may differ greatly between individuals and according to their immunologic state. Typically, about 1% of T cells are Vδ1⁺ and about 1-5% of Vδ1⁺

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phosphate-buffered Saline	Sigma Aldrich	D8537
MACS buffer	Miltenyi Biotec	130-091-222	supplement with BSA and pre-cool before use
BSA	Miltenyi Biotec	130-091-376	not mandatorily from this supplier
anti-human Vd1 FITC (clone: TS8.2)	Thermo Scientific	TCR2730	not mandatorily from this supplier
anti-human CD3 PerCP (clone: SK7)	BD BioSciences	345766	not mandatorily from this supplier or this fluorochrome
anti-human TCRab PE (clone: T10B9.1A-31)	BD BioSciences	555548	not mandatorily from this supplier or this fluorochrome
anti-human CD4 VioBlue (clone: MT466)	Miltenyi Biotec	130-097-333	not mandatorily from this supplier or this fluorochrome
anti-human CD8 APC-H7 (clone: SK1)	BD BioSciences	641400	not mandatorily from this supplier or this fluorochrome
Anti-FITC MultiSort Kit	Miltenyi Biotec	130-058-701	yields better results than anti-FITC MicroBeads
MS columns	Miltenyi Biotec	130-042-201	pre-cool before use
MiniMACS Separator	Miltenyi Biotec	130-042-102
CD4 Positive Isolation Kit	life technologies	11331D