With our fundamental research, we want to understand the initial activation of the adaptive immune response. Calcium is one of the most widely used intracellular signal messengers. If we decipher the complex calcium activation, we may be able to intervene with a signaling machinery to treat pathological conditions such as autoimmune disease.

Using this method, we were able to identify the key players in the NADP signal in cascade and T cells, such as the NADP producing enzyme and NADP binding protein. Moreover, we were able to show that adhesion dependent calcium microdomains contribute to initial calcium signals independent of T cell receptor stimulation. Our method is the first capable of detecting small, fast, and highly dynamic calcium microdomains in primary T cells, and can be easily adapted to other cell types or stimulations and inhibitions.

Additionally, the combination of two chemical dyes allows the calibration of these calcium microdomains. We believe that the initial calcium microdomains shape the adaptive immune response by controlling the fate of individual T cells. Therefore, we want to link the initial calcium response to downstream cycling events and adapt our imaging to live cell super resolution to resolve initial calcium nanodomains.

To begin, take CD4 T cells isolated from mouse lymph nodes and spleen. Centrifuge two to five million cells for five minutes at 300G. Discard the supernatant and resuspend the pellet in 480 microliters of T cell medium containing 10 micromolar Fluo-4 AM and 20 micromolar FuraRed 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. To begin, place 10 microliters of the loaded CD4 positive cells on the prepared slide and let them attach for three to five minutes. Gently add 80 microliters of calcium measuring buffer to the slide.

Select the 100x oil immersion lens. Apply a small drop of immersion oil and place the slide on the microscope stage. Then adjust the focus in brightfield mode.

Select a field of view with up to 10 cells that are not in contact with each other and acquire an image. Next, add 10 microliters of antibody coated beads or stimulant after one minute, and measure for a total of three minutes using 40 frames per second or the maximum frame rate possible. Use the slider to pinpoint the time of contact between a bead and a cell of interest.

In the Options menu on the right hand side, select bead contact:x, y, t. Select the spot on the left side of the image where the cell and bead make contact. Select choose cell by clicking a point inside.

Click on the cell stimulated by this bead preferably in the center. Click on ADD bead contact. Once all bead contacts are defined, click on the Continue button and proceed with additional files.

While type cells displayed rapid calcium microdomain formation within the first second after stimulation, which expanded throughout the cell over the subsequent 15 seconds. Conversely, P2x4 and P2x7 mutant cells showed decreased calcium microdomain formation with P2x4 mutant cells also presenting a lower basal level prior to stimulation. Like murine T cells, calcium microdomains were observed at the bead contact site in human CD4 T cells.