Ldh cytotoxicity assay kit

Toxicity to immortalized human keratinocytes (HaCaTs) was assessed using an LDH cytotoxicity assay kit (G-Biosciences, St. Louis, MO, USA) as described previously^{52 (link)}. Briefly, cells were standardized to 4 × 10⁴ and incubated in 96-well plates for 48 h to allow for seeding, after which media was replaced and treatments were added in serial dilution from 256 to 32 µg/mL. Plates were subsequently incubated for 24 more hours and processed according to the manufacturer’s protocol for chemical induced cytotoxicity.

Atropine, Carbachol, FAEE, Taurolithocholic acid disodium salt, Bovine serum albumin, and Collagenase IV from Clostridium histolyticum were obtained from Sigma-Aldrich (St. Louis, USA); Lipopolysaccharide from Escherichia was purchased from Sigma-Aldrich (Rehovot, Israel). The substrates BOC-Gln-Ala-Arg-7AMC and Z-Arg-Arg-7AMC hydrochloride were obtained from Sigma-Aldrich (Buchs, Switzerland), LDH cytotoxicity assay kit was obtained from G-Biosciences (St. Louis, USA). Hisep LSM 1077, Dulbeccos modified eagle medium, Penstrep, Phosphate buffered saline, Fetal bovine serum were purchased from Himedia laboratories (Mumbai, India). Cytometric bead array (CBA) human inflammatory cytokines kit was purchased from BD Biosciences, USA. Autozyme, an amylase quantification kit was purchased from Accurex Biomedicals (Thane, India); recombinant TNF-α was purchased from Peprotech (Rocky Hill, NJ USA). Polyclonal primary antibodies (anti IL-6, anti-TNF-α, anti-LC3, anti caspase-3) were purchased from Abcam (Cambridge, UK), while anti-amylase antibody was purchased from Santacruz (California, USA). All other chemicals were obtained either from Sigma-Aldrich (Germany, USA) or SDFCL Fine chemicals limited (Mumbai, India).