Barcoding T Cell Calcium Response Diversity with Methods for Automated and Accurate Analysis of Cell Signals (MAAACS)
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).