Naadp am

CD8⁺ T cells from healthy donors were isolated and transfected with siRNA or mRNA as described above. Cells were harvested at a density of 1 × 10⁶/mL and labeled with Indo-1 AM (5 μmol/L; catalog no. 565879, BD Pharmingen) at 37 °C for 30 minutes. Cells were washed with complete RPMI and incubated in complete RPMI for another 30 minutes at 37 °C. Finally, cells were suspended and kept in 37°C water bath before acquisition on an LSR II cytometer (BD Biosciences) running FACSDiva v6.1.3 software (RRID:SCR_001456). After baseline acquisition for 1 minute, the analogue of nicotinic acid, adenine dinucleotide phosphate (NAADP-AM; 10 μmol/L; catalog no. 21000, AAT Bioquest) was added and sample collection immediately resumed. In addition, we used trans-Ned-19 (an inhibitor of NAADP pathway; 10 μmol/L, Enzo Life Sciences, catalog no. ALX-270–503-M001) or Nigericin (an ionophore agent that depletes acidic Ca²⁺ stores; 10 μmol/L, Sigma, catalog no. SML1779) in culture medium 30 minutes before the addition of NAADP-AM to test effects on calcium release at basal level and induced by NAADP-AM. Flow data was analyzed by FlowJo, using the kinetic analyzer function (FlowJo, LLC).

The following antibodies were used for cell stimulation: purified anti-mouse CD38 (Clone 90; BioLegend, 102702), purified rat IgG2a, κ isotype control (Clone RTK2758; BioLegend, 400565). In certain experiments, the following reagents/inhibitors were used at the indicated concentration in cell culture: LPS (E. Coli Serotype R515; Enzo Life Sciences, ALX-581-007-L001), NAADP-AM (3 μM; AAT Bioquest, 21000), GSK2578215A (LRRK2 inhibitor, 1 µM; Tocris Bioscience, 4629), GPN (200 µM; Santa Cruz Biotechnology, sc-252858), EDTA (2 mM; Sigma-Aldrich, E6758), thapsigargin (500 nM; Tocris Bioscience, 1138), 8-Br-cADPR (100 µM; BIOLOG Life Science Institute, B065), 8-Br-ADPR (100 µM; BIOLOG Life Science Institute, B051), Ned-19 (100 µM; Cayman Chemical, 17527), cyclosporine (10 µM; R&D Systems, 1101/100), FK506 (5 nM; Eton Bioscience Inc, 1100060052), CHIR 99021 (GSK3B inhibitor, 5 µM; Tocris Bioscience, 4423), BAPTA-AM (10 µM; R&D Systems, 2787), EGTA-AM (10 µM; AnaSpec Inc, AS-84100), GSK2606414 (EIF2AK3/PERK inhibitor, 10 µM; Tocris Bioscience, 5107), and dorsomorphin (5 µM; Tocris Bioscience, 3093).