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

  1. Use CD45.2+ H2kb+ C57BL/6 wild type (WT) mice as donors for T-cells.
  2. 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 euthanasia if necessary.
  3. Clean the fur and skin of the mouse thoroughly with 70% ethanol. Excise spleens and lymph nodes and separate them into a single cell suspension of splenocytes using a syringe plunger and 40 µm mesh strainers. Wash the strainer and syringe plunger with 1% RPMI (1% FBS containing RPMI media) to collect all splenocytes.
  4. Centrifuge the cell suspension at 800 x g for 5 min at 4 °C. Add 5 mL of ACK lysing buffer (1 mM Na2EDTA, 10 mM KHCO3, 144 mM NH4Cl, pH 7.2) after discarding the supernatant. Incubate the cell suspension for 5 min at room temperature.
    NOTE: ACK lysis buffer is used for lysing red blood cells.
  5. Add 5 mL of 1% RPMI to stop lysis. Centrifuge at 800 x g for 5 min at 4 °C. Discard the supernatant.
  6. Prepare ice-cold magnetic-activated cell sorting (MACS) buffer (0.5% BSA, 2 mM EDTA in PBS, pH 7.2). Degas the buffer before use. Resuspend the cell pellets in 5 mL of MACS buffer.
  7. Count the splenocytes and check for the live and dead cells using a hemocytometer and 1% trypan blue. Save an aliquot of 2 x 106 cells to evaluate the purification yield with flow cytometry analysis.
  8. Resuspend the splenocytes at the concentration of 200 x 106/mL in MACS buffer. Add 0.03 µL of biotin anti-mouse-Ter-119, 0.03 µL of biotin anti-mouse-CD11b, 0.03 µL of biotin anti-mouse-CD45R, and 0.03 µL of biotin anti-mouse-DX5 per 106 cells. Incubate for 15 min at 4 °C.
    NOTE: Biotin anti-mouse-Ter-119, biotin anti-mouse-CD11b, biotin anti-mouse-CD45R, and biotin anti-mouse-DX5 were used to react with erythroid, granulocytes, B-cells, and NK cells respectively. Therefore, these cell subsets are depleted in following step.
  9. Add 10 mL of ice-cold MACS buffer to the cell suspension. Centrifuge at 800 x g for 5 min at 4 °C. Discard the supernatant.
  10. Resuspend the cell pellets in the MACS buffer at a concentration of 100 x 106/mL. Add anti-biotin microbeads (0.22 µL/106 cells) to the splenocyte suspension. Mix well and incubate for an additional 15 min at 4 °C. Wash the cell suspension once with 10 mL of ice-cold MACS buffer. Centrifuge at 800 x g for 5 min at 4 °C and discard the supernatant.
  11. Put a magnetic separating column in the magnetic field. Rinse the column with 3 mL of MACS buffer. Drop the cell suspension onto the column. Collect the flow-through consisting of unbound, enriched T-cells, in a new 15 mL conical tube. Wash the MS column with 3 mL of ice-cold MACS buffer.
    NOTE: Ensure that the column is empty prior to performing the washing steps.
  12. Centrifuge the cell suspension at 800 x g for 5 min at 4 °C. Resuspend the cell pellet in 5 mL of MACS buffer.
  13. Count the cells in 1% trypan blue using a hemocytometer. Save an aliquot of 2 x 106 cells for a purity check by flow cytometry.
    NOTE: The average yield of splenic T-cells isolated by this method is ~20−25 x 106 cells per mouse.

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

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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).

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