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

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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⁺ H2k^b+ 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.

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Материалы

Name	Company	Catalog Number	Comments
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