<p>The fluorescence intensity of all loaded and responsive murine 3A9 naive CD4+ T cell acquired by confocal microscopy and detected by MAAACS are normalized and displayed as a barcoded response over time (color-coded as indicated). <b>Anti TCR antibody coated surface (left panel):</b> Cells were seeded onto anti TCR antibody coated Lab-tek chambers with (n<sub>cells</sub> = 117) or without 2-APB (n<sub>cells</sub> = 276). <b>Antigen presenting cell (middle and right panel):</b> Cells were seeded onto COS-7 experimental antigen presenting cells loaded or not with HEL peptides (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003245#s4" target="_blank">materials and methods</a>) in the presence of 2-APB (n<sub>cells</sub> = 266, without peptide; n<sub>cells</sub> = 112, with peptide) or without 2-APB (n<sub>cells</sub> = 491, without peptide; n<sub>cells</sub> = 349 with peptide). <b>(B) Calcium response modes in naive CD4+ T cells</b> represented in pie charts (unactivated in gray, maintained in pink, oscillating in green and unique in yellow). <b>(C) Naive CD4+ T cell calcium responses upon different stimuli</b> revealed by MAAACS. Left panel - fluorescence amplitude of T cells in the absence or presence of 2-APB. Middle panel - The response fraction of T cells in the absence or presence of 2-APB. Right panel - frequency of calcium bursts in T cell in the absence or presence of 2-APB. Scatter plots only show signals from responding cells (TH<sub>act</sub> = 1.74 without 2-APB, TH<sub>act</sub> = 1.89 with 2-APB). The mean value +/− SEM is represented in red. Statistical tests were carried out with the Mann Whitney non-parametric test (*** = p<0.001; ** = 0.0010.).</p
