To begin, prepare a mouse splenic single cell suspension with one times 10 to the power of eight cells per milliliter in a volume of 0.1 to two milliliters. Add 50 microliters of rat serum to the sample. Transfer the sample to a five milliliter polystyrene round bottom tube.
Add 50 microliters of isolation cocktail to the sample. Mix well and incubate for 7.5 minutes at room temperature. Now add 50 microliters per milliliter of depletion cocktail.
Mix well and incubate for 2.5 minutes. Meanwhile, vortex the magnetic beads to ensure even dispersion. Add 75 microliters of the magnetic beads to the sample.
Mix well and incubate for 2.5 minutes. Top up the sample with RPMI 1640 medium to a final volume of 2.5 milliliters and mix several times. Then place the tube into the magnet for 2.5 minutes.
Pick up the magnet and invert it with the tube in one continuous motion, pouring the enriched cell suspension into a new tube. Determine the cell number using a hemocytometer. Perform flow cytometric analysis for naive CD four positive T-cell before and after isolation.
After gating, transfer the cell suspension to a new centrifuge tube. Centrifuge at 400G for five minutes and discard the supernatant. Resuspend the pellet in 200 to 500 microliters of RPMI 1640 medium supplemented with 10%FBS.
For each one milliliter of cell culture, add two microliters of leukocyte activation cocktail and mix. Culture the cells in a carbon dioxide incubator at 37 degrees Celsius with saturated humidity for four to six hours.
