Bromoenol lactone

Resorufin, Resorufin sodium salt, naphthofluorescein, palmitoyl chloride, phospholipase A₂ from porcine pancreas, arachidonyl trifluoromethyl ketone, bromoenol lactone, ionomycin, and H₂O₂ 30% (w/w) were purchased from Sigma-Aldrich. DDAO, arachidonoyl chloride, and LY311727 were purchased from Santa Cruz Biotechnology. 7-HCA was purchased from Cayman Chemical. Syto 9 was purchased from Life Technologies. Egg phosphatidylcholine was purchased from Avanti Polar Lipids, Inc.

2 μg/mL human recombinant cPLA2 protein (#6659-PL-010, R&D systems) was mixed with substrates containing 10 μM LacCer (Cat. #1507, Matreya) or vehicle and the fluorescence signal was read after 10 minutes room temperature incubation. For in vitro cPLA2 enzymatic activity assay, 5,000,000 cells were seeded on a 10-cm dish and kept rested for 2 days, pretreated with 10 μM LacCer or 10 μM LacCer + 500 nM cPLA2i (#sc-296161, Santa Cruz Biotechnology) for 30 mins, stimulated for 1 hour with 10 ng/ml TNFα (#410-MT-010, R&D Systems) and 100 ng/ml IFNγ (#485-MI-100, R&D Systems). After stimulation, the astrocytes were lysed with 250 μL of RIPA buffer supplied with protease and phosphatase inhibitor mix, treated with 5 μM iPLA2 inhibitor (Bromoenol Lactone, #B1552, Sigma) at 25°C for 15 minutes, and then mix 50 μL of cell lysate and 50 μL of substrate mixture supplied in EnzChek™ Phospholipase A2 Assay Kit (#E10217, Thermo Fisher Scientific) for detecting cPLA2 activity. After 10 mins incubation at R.T., the fluorescence signal was read by Infinite M1000 PRO Microplate Reader (Tecan) and the procedures are as suggested by the manufacturer.