Cells were incubated at a concentration of 0.5×107per selleck mL with 5 μM Indo-1AM (Invitrogen, Molecular Probes) for 60 min at 37°C, stained with
anti-CD8α-PE for 10 min and left at room temperature in the dark. The viability of cells after Indo-1AM loading was >90% as assessed by propidium iodide staining gated on the lymphocyte FSC/SSC population. Prior to data acquisition, the cell suspensions were warmed to 37°C in the dark for 10 min and then aliquoted in 200 μL, then CaCl2 was added to a final concentration of 1 mM and Ca2+-flux was measured with a LSRII (BD) cytometer equipped with a 355 nm UV laser at 37°C using a custom-built heating device adapted to cytometer tubes. After acquisition of the baseline levels for 60 s, anti-CD3 or anti-γδ TCR mAb was added and the cross-linking anti-Hamster Ab were added at second 90. The following concentrations of mAb were used: systemic T-cell compartment, 100 μg/mL of anti-CD3 (clone 145-2C11) with 180 μg/mL of anti-hamster and 100 μg/mL of anti-γδ TCR (clone GL3) with 180 μg/mL of anti-hamster final concentrations;
iIEL compartment, 200 μg/mL of anti-CD3 with 180 μg/mL anti-hamster and 100 μg/mL of anti-γδ TCR (clone GL3) with 360 μg/mL of anti-hamster final concentrations. After the stimulation, the cells were acquired for additional 3 min. Ionomycin was used as a positive control for Ca2+-flux (2 μg/mL). The kinetic Ca2+ changes were analyzed in MLN0128 FlowJo software (Version 8.8.2, Treestar). For cytokine quantification, C57BL/6 iIEL were incubated in 96-well plates coated either with 10 μg/mL of anti-γδ TCR (clone GL3 and GL4), anti-αβ TCR (clone H57-597) or anti-CD3 (clone 145-2C11) for a period of 24 h and the supernatants were analyzed for CCL4 and IFN-γ by cytometric bead array (CBA, BD Biosciences) according to the manufacturer’s instructions. For intracellular cytokine detection in iIEL populations, WT C57BL/6 iIEL
were incubated in a 24-well plate coated with 10 μg/mL of anti-γδ TCR (clone GL3 or GL4), anti-αβ TCR (clone H57-597), anti-CD3 (clone 145-2C11) or in presence of PMA (10 ng/mL) and ionomycin (2 μg/mL), for 4 h. Brefeldin A (10 μg/mL) was added for the last 3 h. The cells were stained with surface marker and intracellular cytokine antibodies for FACS analysis of CCL4, IL-17A and IFN-γ. FACS experiments were performed on an LSRII Farnesyltransferase flow cytometer (BD Biosciences) and the data were analyzed by FlowJo software (Version 8.8.2, Treestar). All bar graphs are presented as mean±SEM and were made using GraphPad Prism software (Version 4.03). Fold changes of Violet/Blue ratio were obtained by dividing the peak values (after antibody Ca2+-flux induction either with clones 145-2C11 or GL3) with the mean baseline levels (before antibody Ca2+-flux induction). These values obtained from iIEL or systemic T cells in PBS (control group) and anti-γδ TCR (GL3 group) treated mice conditions were compared using unpaired one-tailed t test.