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T Cells Isolation and Purification

Remove the spleen.

Grind the spleen with the flat end of a syringe in 5 ml of RPMI on a 100 mm culture dish.

Pass through all cells through a cell strainer into a 50 ml tube. Wash the cell culture dish with 5 ml of RPMI twice and combine all cells.

Spin down the cells and remove the supernatant. Re-suspend the cell pellet into 5 ml of red blood cell lysis buffer followed by incubation at RT for 2 min. Dilute cells with 45 ml of RPMI or 1X PBS.

Spin down cells and re-suspend the cell pellet in 1 ml of RPMI/10% FCS. Incubate the cells at RT for 30 min.

Purify T cells with a sterile Nylon column. Wet the column with 5 ml of RPMI / 10% FCS followed by another 5 ml wash. Leave 1 ml of medium on the top of the column.

Incubate the column at 37oC for ~ 1 hour.

Open stop cock and allow media to run through column. Add 2 ml media and allow this to run through the column. Add 1 ml of splenocytes (1-2 x 108 cells). Allow cells to enter column. Add another 1 ml of media and allow to enter column. Cover the column with 1 ml media and incubate at 37oC for one hour.

Collect the T cells by washing column with 10 ml of media. Do not plunge, as this removes B cells.

Spin and re-suspend the cells and count them.

To 1 ml of cells (1-6 x107) add 8 µl (8 µg) of anti-MHCII and / or 8 µl (4 µg) of anti-CD8.

Incubate on ice for 30 min. Add 9 ml of PBS or RPMI. Spin and re-suspend the cells into 1 ml of complement. Incubate for 30 min at 37oC. Commercially available complement reagent is prepared by re-suspending the lyophilyzed pellet in 1 ml of cold H2O. This should be diluted in 9 ml of RPMI.

After incubation, add 9 ml of RPMI or PBS. Repeat antibody-complement cycle once more. Wash the cells with RPMI and count.

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