Figure 1.
BD PBX, a highly sensitive calcium probe allows ratiometric analysis and antibody labeling of primary cells. (A) Comparison of excitation and emission spectra of Fluo-4-AM and the calcium indicator included in the PBX calcium assay kit, BD PBX.
The excitation and emission intensity of Fluo-4 AM was exported from http://probes.invitrogen.com/media/spectra/data/14200ca.txt. To measure the spectrum of BD PBX, 3A9 hybridoma cells were loaded and analyzed with a spectrofluorimeter. Excitation intensity was measured for a fixed emission wavelength at 516 nm. Emission intensity was measured for a fixed excitation wavelength at 495 nm (B) In vitro calibration of the BD PBX and Fluo-4 AM. 3A9 T cells loaded with Fluo-4 AM or BD PBX as indicated in Materials and Methods, were washed three times in calcium free medium from the calcium calibration kit. Fluorescence of the cells was measured on a spectrofluorimeter from 500 nm to 650 nm. By a gradual increase of extracellular calcium concentration allow to plot extracellular calcium concentration as a function of the (F-Fmin)/(Fmax-F) ratio enabling to determine the Kd value. (C). Comparison of calcium responses using Indo-1 or BD PBX upon various doses of calcium ionophore ionomycin. 3A9 T cells were loaded with Indo-1 or BD PBX as indicated in Materials and Methods. Cells were stimulated 1 min after the start point with different concentration of ionomycin and fluorescence acquisitions were performed with a LSR I flow cytometer at 37°C. Fluorescence amplitudes or Indo-1 ratio were calibrated using the estimated in situ Kd of the Fluo-4 AM (1 µM) or Indo-1 AM (0.23 µM) with a scale factor of 2.9. (D) Comparison of calcium responses using Indo-1 or BD PBX upon thapsigargin or anti-TCR crosslinking. 3A9 T cells were loaded with Indo-1 or BD PBX as indicated in Materials and Methods. Cells were stimulated 1 min after the start point (red arrow) with thapsigargin, or a complex of antibody against CD3-biotin/streptavidin. Data were normalized to the baseline before stimulation to allow comparison between recordings. (E) Calcium response upon thapsigargin stimulation of purified mouse CD4+ T lymphocytes. Mouse CD4+ T lymphocytes were purified from spleen and lymph nodes and overnight serum starved. Cells were loaded with BD PBX and analyzed by flow cytometry under thapsigargin stimulation (red arrow in the right panel) with (red) or without (black) cell surface labeling with an anti CD4 coupled to APC antibody (left panel).
Figure 2.
Calcium signal processing. (A) Automatic tracking of high density moving cells by MAAACS.
Raw fluorescence experimental images (displayed with arbitrary pseudo-colors) are first filtered using a median filter to eliminate electronic noise emanating from PMT detection. The filtered image is correlated with a cylindrical mask (depicted next to the central arrow) to detect cells which are represented by Gaussian peaks keeping the overall initial intensities (height). Note that the width of the mask is set to adequately match the average cell width. Cell positions are finally linked from frame to frame following the MTT scheme to obtain trajectories that are color coded according to fluorescence intensity. Scale bar, 20 µm. The lower panel depicts the 2.5D views of the cropped areas. (B) Automatic signal analysis. Raw fluorescence intensities are normalized by taking into account the basal fluorescence enabling comparisons of the calcium signals between cells. The central panel represents the fluorescence intensity distribution before the maximal amplitude (gray area in the left panel). The median value (24.7 in this example, red dotted line in the left panel) is defined as the baseline for the cell. Raw intensities are thus normalized to this value (right panel). The calcium flux in each individual cell can be characterized by several analytical parameters concerned with its intensity (fluorescence amplitude and maximal amplitude) and its shape (response fraction, shown as a ratio of pink and blue areas, and number of bursts/min shown as green lines on the right panel). (C) Graphical representations of the analytical parameters extracted from a movie treated with MAAACS. The global response of cells is represented with a barcode view: the fluorescence amplitude of each cell is plotted along a horizontal line as a function of the time with a color coded intensity (dark to blue below the threshold of activation and yellow to red above the threshold of activation. Threshold of activation is set at 2 here). All analytical parameters (fluorescence amplitude, response fraction and number of bursts/min) extracted from the analysis with MAAACS are plotted in a scatter plot with the mean (+/−SEM, shown in red) and a pie-chart represents the cell response heterogeneity (inactive, maintained, oscillating and unique) (ncells = 55).
Figure 3.
MAAACS tracking performance versus exhaustive manual tracking. (A) Overlay of cell trajectories analyzed manually or with MAAACS.
Traces resulting from manual and automatic tracking (with MAAACS) are plotted together (respectively in orange and gray) to evaluate the tracking efficiency either for primary T cell or T cell hybridomas. (B) Comparison of MAAACS tracking performances versus manual tracking. Left panel - Each trajectory obtained with MAAACS is compared to the corresponding manual trace and the percentage of overlap is represented in a scatter plot for different cell types (primary T cells and T cell hybridomas) with or without reconnection (recon.). Right panel – For each MAAACS trajectory with reconnection, the average number of trace fragments are plotted (+/− SEM). (C) Reconnection algorithm of MAAACS aborted traces. An algorithm was implemented to reconnect trace fragments generated with the automatic tracking. Traces terminating before the end of acquisition (gray trace) were identified by the algorithm. Candidates for reconnection were selected among non-overlapping traces (blue and red traces, but not the green trace, starting before the end of the gray trace) following two additional criteria, narrow distance between traces and similar raw fluorescence amplitudes. The final decision is made using the Graphical User Interface (GUI). (D) Impact of the reconnection on the trajectory overlap between manual tracking and MAAACS. Scatter plot of the percentage of trajectory overlap with or without reconnection supervision calculated for each trace. The average (+/− SEM) are plotted in red.
Figure 4.
2-APB induced inhibition of CRAC channel activity prior to thapsigargin stimulation evaluated using flow cytometry as compared to confocal imaging analysis using MAAACS. (A) Intracellular calcium mobilization measured in HBSS without calcium and magnesium or by using the CRAC channel inhibitor 2-APB.
3A9 loaded T cells were resuspended in HBSS Hepes without Ca2+/Mg2+ or in HBSS Hepes with 2-APB before stimulation with thapsigargin (red arrow) and analyzed by flow cytometry. Cell fluorescence is represented on a pseudo-colored dot plot. (B) Global and intracellular calcium mobilization evaluated by flow cytometry. 3A9 loaded cells were stimulated with thapsigargin in the absence (−2-APB) or in the presence (+2-APB) of 2-APB. Evolution of the normalized median of fluorescence of the population is plotted vs. time. (C) Global and intracellular calcium mobilization evaluated by confocal microscopy using MAAACS for detection and analysis. 3A9 loaded cells were stimulated with thapsigargin (red arrow) in the absence (−2-APB) or in the presence (+2-APB) of 2-APB and fluorescence was monitored for 35 min under a confocal microscope. Videos were subjected to MAAACS analysis, and the average fluorescence of the tracked cells is plotted across time (left panel). For better comparison with flow cytometry, images from the first 10 minutes of recording are zoomed in (right panel). (D) Analytical parameters of calcium signaling under thapsigargin stimulation in the absence or presence of 2-APB. The fluorescence amplitude, the response fraction and the number of bursts/min are analyzed as a scatter plot (ncells = 104, −2-APB; ncells = 111, +2-APB). The mean value +/− SEM is represented in red in each condition. Statistical tests were carried out using the Mann Whitney non-parametric test (*** = p<0.001; ** = 0.001<p<0.01; * = 0.01<p<0.05; ns = p>0.).
Figure 5.
Evaluation of calcium response of a heterogeneous population of individual T cell hybridomas upon different experimental stimuli.
(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).
Figure 6.
Effect of peptide concentration on the ER release of calcium.
COS-7 cells expressing MHC class II molecules I-AK were loaded overnight with increasing concentrations of HEL 46–61 peptides. Calcium responses in 3A9 hybridomas with 2-APB were analyzed with MAAACS. (A) Percentage of responding cells for various peptide concentrations was obtained with activation thresholds (THact) respectively for hybridomas stimulated with increasing doses of 1.4 (0.5 nM), 1.8 (5 nM), 1.7 (50 nM), 1.6 (500 nM), 1.5 (5 µM) with 2-APB. (B) Fluorescence amplitude of calcium response of activated cells as a function of peptide concentration is represented on a scatter plot. (C) Response fraction of activated cells as a function of peptide concentration is represented on a scatter plot. (D) Frequency calcium response bursts of activated cells as a function of peptide concentration is represented on a scatter plot. The mean value +/− SEM is represented in red. Statistical tests were carried out using Mann Whitney's non-parametric test (*** = p<0.001; ** = 0.001<p<0.01; * = 0.01<p<0.05; ns = p>0.) (E) Mode of calcium response: For each antigenic peptide concentration, modes of calcium response are represented as a pie chart: unactivated in gray, maintained in pink, oscillating in green and unique in yellow.
Figure 7.
Naive CD4+ T cells display mainly intracellular calcium oscillations upon antigenic challenge. (A) Barcoding of stimulation.
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). Anti TCR antibody coated surface (left panel): Cells were seeded onto anti TCR antibody coated Lab-tek chambers with (ncells = 117) or without 2-APB (ncells = 276). Antigen presenting cell (middle and right panel): Cells were seeded onto COS-7 experimental antigen presenting cells loaded or not with HEL peptides (see materials and methods) in the presence of 2-APB (ncells = 266, without peptide; ncells = 112, with peptide) or without 2-APB (ncells = 491, without peptide; ncells = 349 with peptide). (B) Calcium response modes in naive CD4+ T cells represented in pie charts (unactivated in gray, maintained in pink, oscillating in green and unique in yellow). (C) Naive CD4+ T cell calcium responses upon different stimuli 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 (THact = 1.74 without 2-APB, THact = 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; ** = 0.001<p<0.01; * = 0.01<p<0.05; ns = p>0.).