Selective Isolation of CD4+ Vδ1+ γδ T Cells from Human Peripheral Blood Mononuclear Cells
Published: August 31, 2023
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 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 b…
Divulgazioni
The authors have nothing to disclose.
Materials
| 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 |
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Citazione di questo articolo
Selective Isolation of CD4+ Vδ1+ γδ T Cells from Human Peripheral Blood Mononuclear Cells. J. Vis. Exp. (Pending Publication), e21563, doi: (2023).