Detecting Ca²⁺ Microdomains on a Subcellular Level in Primary Murine T Cells Using Fluorescence Microscopy

To begin, place 10 microliters of the loaded CD4+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 Wild-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.