Phospholipase d activity assay kit

Cell culture medium RPMI1640, fetal calf serum (FCS), antibiotics, and trypsin were from Invitrogen (MA, USA). Autotaxin inhibitor (PF8380) was from Echelon Biosciences (UT, USA) and the LPA5 antagonist AS2717638 was a gift from Prof. Marc Nazare (Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany). Lipopolysaccharide (LPS) from Escherichia coli (O111:B4), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), and the phospholipase D activity assay kit were from Sigma-Aldrich (MO, USA). ELISA kits were from Peprotech (NJ, USA). RNeasy lipid tissue mini kit was from Qiagen (Hilden, Germany). Antibodies were from Cayman chemicals (MI, USA), Abgent (CA, USA), Merck Millipore (MA, USA), Fujifilm Wako Chemicals USA Inc (VA, USA) Cell Signaling Technology (MA, USA), Abcam (Cambridge, UK), Santa Cruz Biotechnology Inc. (TX, USA), and Agilent Dako (CA, USA), (as listed in Table 1). Primers were from Qiagen (Germany) and Invitrogen (MA, USA) (listed in Table 2). Total nitric oxide assay kit was from Enzo Life Sciences, Switzerland. Lactate dehydrogenase (LDH) activity kit was from Cayman Chemicals (MI, USA).

Lipase and phospholipase assays using the purified human EPDR1 were performed according to the manufacturers’ protocols. EPDR1 did not show any activities using the Lipase Activity Assay Kit (catalog No. MAK046; Sigma–Aldrich, St Louis, Missouri, USA), the Phospholipase D Activity Assay Kit (catalog No. MAK137; Sigma–Aldrich) or the EnzChek Direct Phospholipase C Assay Kit (catalog No. E10215; Thermo Fisher Scientific, Waltham, Massachusetts, USA).

Enzyme activity was measured using the Phospholipase D Activity Assay Kit (Sigma). In the used assay, phospholipase D (PLD) hydrolyzed PC to choline, which was determined using choline oxidase, resulting in a colorimetric (570 nm) product proportional to the PLD activity in the sample. Unit definition: one unit of PLD catalyzes the formation of 1 mM of choline per minute under the assay conditions (pH 7.4). Detailed analysis was carried out according to the manufacturer’s instructions. Extractions for analyzing PLD activity were performed by tobacco BY-2 cell (100 mg) homogenization in Cellytic buffer (Sigma) and then centrifugation at 4500× g for 5 min. The obtained supernatants were immediately tested. The calibration curve was prepared according to the manufacturer’s information for colorimetric detection. The PLD activity was measured on the 4th, 5th and 7th days of the experiment—4, 24 and 72 h after lead administration, respectively.