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…
Disclosures
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 |
Cite This Article
T-Cell Enrichment: A Technique to Isolate T-Cells from Mixed Cell Population by Magnetic Separation. J. Vis. Exp. (Pending Publication), e20269, doi: (2023).