T-Cell Enrichment: A Technique to Isolate T-Cells from Mixed Cell Population by Magnetic Separation

Published: April 30, 2023

Abstract

Source: Nguyen, H. D., et al. Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease. J. Vis. Exp. (2020).

This video describes T-cells enrichment from the spleen of a mouse by negative selection using a magnetic separator. The technique involves targeting the other cells in the sample for depletion using antibodies or ligands directed against specific cell surface antigens.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Day 1: Prepare T-cells from the donor mice Use CD45.2+ H2kb+ C57BL/6 wild type (WT) mice as donors for T-cells. Euthanize C57BL/6 mice by carbon dioxide (CO2) asphyxiation and wait for 2−3 min until they are unconscious. Perform cervical dislocation as a secondary euthan…

Divulgaciones

The authors have nothing to disclose.

Materials

RPMI 1640  Thermo Fisher Scienctific  11875-093  Media
MidiMACS Miltenyi Biotec  130-042-302  T-cell enrichment
0.5 M EDTA  pH 8.0 100ML  Fisher Scientific  BP2482100  MACS buffer
10X PBS Fisher Scientific  BP3994  MACS buffer
Anti-Biotin MicroBeads Miltenyi Biotec  130-090-485   T-cell enrichment
Anti-Human/Mouse CD45R (B220)  Thermo Fisher Scientific  13-0452-85   T-cell enrichment
Anti-Mouse CD4 Biotin  Thermo Fisher Scientific  13-0041-86  T-cell enrichment
CD11b Thermo Fisher Scientific  13-0112-85   T-cell enrichment
CD25-biotin Thermo Fisher Scientific  13-0251-82   T-cell enrichment
CD45R Thermo Fisher Scientific  13-0452-82  T-cell enrichment
CD49b  Monoclonal Antibody (DX5)-biotin Thermo Fisher Scientific  13-5971-82  T-cell enrichment
Anti-Mouse TER-119 Biotin  Thermo Fisher Scientific  13-5921-85   T-cell enrichment
Anti-mouse CD45.1 PE  Thermo Fisher Scientific  12-0900-83 Flow cytometry analysis
C57BL/6 mice  Charles River 27 Donors/Recipients
Flow cytometry tubes Fisher Scientific 352008 Flow cytometry analysis
Cell strainer 40 uM Thermo Fisher Scientific  22363547 Cell preparation

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Citar este artículo
T-Cell Enrichment: A Technique to Isolate T-Cells from Mixed Cell Population by Magnetic Separation. J. Vis. Exp. (Pending Publication), e20269, doi: (2023).

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