Skip to main content
. 2013 Sep 26;9(9):e1003245. doi: 10.1371/journal.pcbi.1003245

Figure 5. Evaluation of calcium response of a heterogeneous population of individual T cell hybridomas upon different experimental stimuli.

Figure 5

(A) Barcoding of stimulation. The fluorescence intensity of all loaded and responsive (except for negative control conditions, i.e. anti CD45 coated surface) 3A9 T cell hybridomas acquired by confocal microscopy and detected by MAAACS are normalized and displayed as a barcoded response over time (color-coded as indicated). Anti CD45 antibody coated surface (left panel): Cells were seeded onto anti CD45 antibody coated Lab-tek chambers in the presence or absence of 2-APB. All tracked cells are represented on the barcode. Anti TCR antibody coated surface (middle panel): Cells were seeded onto anti TCR antibody coated Lab-tek chambers with or without 2-APB. Antigen presenting cell (right panel): Cells were seeded onto COS-7 experimental antigen presenting cells (see materials and methods) with or without 2-APB. (B) Mode of calcium response: To compare the calcium signal among different populations of individual cells with different types of stimuli we classified cells into four classes according to the intensity, number of bursts and response fraction. For each stimulus, these are represented as a pie chart: unactivated in gray, maintained in pink, oscillating in green and unique in yellow (C) Analytical parameters of calcium signaling for different stimuli (anti TCR antibody coated surface and antigen presenting cells): fluorescence amplitude, response fraction and bursts/min. The mean value (+/− SEM) of each analytical parameter is shown for each activated cell as a dot on each scatter plot. A non-parametric two-tailed unpaired Mann-Whitney test was used. *** indicates a p-value<0.0001. Activation thresholds (THact) were 1.7 for hybridomas stimulated with antibody without 2-APB, 1.92 with 2-APB, 1.80 for APC without 2-APB and 1.62 without 2-APB) (ncells = 146, anti CD45 − 2-APB; ncells = 96, anti CD45 + 2-APB ncells = 128, anti TCR − 2-APB; ncells = 152, anti TCR + 2-APB; ncells = 209, APC − 2-APB; ncells = 515, APC + 2-APB).