Lsri flow cytometer

Calcium flux analyses of human T-cells were performed according to^{67 (link)}. Briefly, 3 × 10⁵ cells/mL in phenol red-free RPMI 1640 (Life Technologies) containing 10% FCS were loaded with 5 µg/mL Indo-1 AM (MoBiTec, Göttingen, Germany) at 37 °C for 45 min, followed by an additional incubation for 45 min in medium without Indo-1. Cells were kept on ice before equilibration at 37 °C for 5 min, directly before measurement. Changes in intracellular calcium were monitored using a flow cytometer LSRI (BD Biosciences, Heidelberg, Germany). Cells were illuminated using the 325 nm laser-line of a helium-cadmium laser. Fluorescence emissions at 405/30 nm (calcium-bound Indo) and 510/20 nm (free Indo) were detected simultaneously, analyzing the ratio of bound to free Indo over time. After monitoring the baseline activity for 1 min, the cells were stimulated by 10 µg/mL CD3 mAb (clone UCHT1, BD Biosciences) and cells were measured for another 6 min. To confirm proper Indo-1 loading, the cells were then treated with 10 µg/mL ionomycin (Sigma-Aldrich). Kinetics were analyzed using FlowJo v7.6.3 software (Tree Star, Ashland, USA).

Beta cells were identified by staining with Newport Green (NG; 10 μmol/L; Invitrogen, Molecular Probes, Eugene, OR), a fluorescent probe that detects zinc in the insulin granules of beta cells [46 (link)]. Damaged and dying islet cells were assessed using 7-Aminoactinomycin (7AAD, 10 μg/ml; Life Technologies, Eugene, OR) or by Sytox green (31.25 nmol/L; Invitrogen, Molecular Probes) uptake (https://dx.doi.org/10.17504/protocols.io.kwwcxfe) [27 (link)]. For intracellular staining, isolated islet cells were fixed in 2% paraformaldehyde (Sigma-Aldrich) and permeabilized using 0.3% saponin (Sigma-Aldrich). The cells were stained with 10E4 mouse anti-human HS mAb (10E4, 1/50; Seikagaku, Tokyo, Japan or US Biological/Amsbio, Abingdon, UK), mouse anti-mouse Col18 mAb (1/50; Santa Cruz Biotechnol., Santa Cruz, USA) or the corresponding isotype control Ig (mouse IgM_κ or IgG_2bκ; BD Biosciences, San Jose, CA) followed by goat anti-mouse Ig-R-phycoerythrin (1/100; Southern Biotech, Birmingham, AL)
(https://dx.doi.org/10.17504/protocols.io.kwzcxf6) [27 (link)]. The geometric mean fluorescence ratio (GMFR) was calculated by dividing the geometric mean fluorescence intensity (GMFI) of cells stained with primary mAb by the GMFI obtained with the relevant isotype control Ig [27 (link)]. Cells were analyzed using a BD LSRI flow cytometer and CellQuest™ Pro software (version 6.0; BD Biosciences).