Fluo 8 calcium dye

Calcium flux was taken as an indication of T-cell activation. To compare the calcium flux in Jurkat cells, fluo-8 calcium dye (Abcam) was used, and the assay was performed as described previously.^{15 (link)} Briefly, 10,000 HFLS-RA cells were seeded in each well in 96 well plates, and after 72 h incubation, HFLS-RA cells were treated with different concentrations of peptide (5 nM and 200 nM) and incubated for 1 h. Simultaneously Jurkat cells were washed three times with PBS and incubated with fluo-8 calcium dye for 1 h, followed by washing of Jurkat cells 3 times. These dye-loaded cells were activated by CD3 antibody, and immediately the Jurkat cells were resuspended in PBS supplemented with calcium chloride and added over the peptide treated HFLS-RA cells. The plate was placed into the fluorescence plate reader (Synergy H1 Hybrid plate reader (Biotek)), and fluorescence reading of the fluo-8 calcium dye (Ex/Em = 490/525 nm) was measured for 2.5 h by using the kinetic parameter. Experiments were repeated three times. A graph of relative change in fluorescence vs. time was plotted. Results were represented as mean ± standard deviation.