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

Protocolo

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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Materiais

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