Loading of Primary Mouse CD4⁺ T Cells and Coating of Protein G Magnetic Beads with Antibodies for Local Ca²⁺ Imaging

To begin, take CD4-positive T cells isolated from mouse lymph nodes and spleen. Centrifuge two to five million cells for five minutes at 300 g. Discard the supernatant and resuspend the pellet in 480 microliters of T cell medium containing 10 micromolar Fluo-4 AM and 20 micromolar Fura Red AM.Cover the Falcon tube with aluminum foil and incubate the cells for 20 minutes at room temperature in the dark.

Then add two milliliters of T cell medium to the cells and continue incubating for an additional 30 minutes. Gently mix Protein G magnetic bead suspension and transfer 12.5 microliters to a new tube. Place the tube on a magnetic stand to allow the beads to migrate to the magnet.

Gently pipette out the storage buffer from the opposite side of the magnet to remove it. To remove any remaining storage buffer, add 500 microliters of PBST and vortex for 10 seconds. Place the tube on the magnetic stand again and remove the buffer.

To coat the beads with antibodies, resuspend them in 7.5 microliters of PBST and add five microliters each of anti-CD3 and anti-CD28. Incubate for 30 to 60 minutes while continuous mixing at room temperature. Wash the coated beads three times with 500 microliters of PBST, followed by a wash with 500 microliters of calcium buffer using the magnetic stand, then resuspend the beads in 200 to 400 microliters of calcium buffer.