Alamarblue assay kit

J774A.1 macrophages were incubated for 5 h with LPS (1 μg/ml) followed by incubation for 0.5 h with CVL (20 μM). Cells were then incubated with ATP (5 mM) for 0.5 h. The cell viability was measured by the AlamarBlue® assay kit according to the instruction manual (AbD Serotec Ltd). The LDH release assay was performed by the CytoTox 96® Non-radioactive Cytotoxicity assay kit according to the instruction manual (Promega, Madison, WI). The membrane integrity was measured by staining the cells with 40 μg/ml of propidium iodide (PI) (Sigma-Aldrich) and analyzed by flow cytometry. The cell size was determined by calculating 20 representative cells using ImageJ software.

Antibodies against PKC-α, PKC-ε and PKC-δ, COX2, p-IKK-α and anti-p-IκB-α were procured from Santa Cruz Biotechnology (Dallas, TX). Antibodies against p-ERK1/2, p-JNK1/2 and p-p38 were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against iNOS and β-actin, lysis buffer and polyvinylidene fluoride (PVDF) membrane were purchased from EMD Millipore (Bedford, MA). LPS (Escherichia coli O111:B4) and protease/phosphatase inhibitor cocktail were purchased from Sigma-Aldrich (St. Louis, MO). Enzyme-linked immunosorbent assay (ELISA) kits and enhanced chemiluminescence (ECL) reagents were purchased from Thermo Fisher Scientific (Waltham, MA). RPMI 1640 was purchased from Gibco Laboratories (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Biological Industries Ltd. (Kibbutz Beit Ha-emek, Israel). L-glutamine was purchased from Life Technologies (Carlsbad, CA, USA). Alamar Blue assay kit was purchased from AbD Serotec (Oxford, UK). QUANTI-Blue medium was purchased from InvivoGen (San Diego, CA).