Lactate dehydrogenase kit

E. coli was exposed to 0, 1, 10, 100, or 1000 μg/mL of MSNs in PBS (pH 7.0). Subsequently, the cells were incubated in PBS at 37°C under constant orbital shaking at 220 rpm for 6 h, after which the cells were separated by centrifugation at 4000 rpm for 5 min and the supernatants transferred into 96-well plates. Lactate dehydrogenase (LDH) activity was determined using a lactate dehydrogenase kit (Sigma-Aldrich, USA), according to the manufacturer’s protocol, with final detection of absorption at 450 nm using a Lambda-25 plate reader (Perkin-Elmer, USA). The results are expressed as the means ± SD of six parallel measurements.

An enzymatic method according to the procedures in the Sigma-Aldrich assay kits were used for the determination of total cholesterol (TC), triglycerides (TGs), and high density lipoprotein cholesterol (HDL) with a BT-3000 auto analyzer (Biotecnica Instruments, Licenza, 18-00156 Rome Italy). Low-density lipoprotein (LDL), cardiac risk ratio (CRR), and atherogenic coefficients (ACs) were calculated as follows [24 (link)]:
Creatine kinase was assayed by spectrophotometry at an absorbance of 450 nm according to the assay protocol described by Horder et al. [25 (link)]. Lactate dehydrogenase was determined by enzymatic colorimetry using the methods described in the Lactate Dehydrogenase Kit (Sigma Aldrich St. Louis, MO, USA, Catalog No-MAK066) while the cardiac troponin test was performed by enzyme-linked immunosorbent assay (ELISA) in accordance with the procedures on the ELISA kit (East Biopharm, Hangzhou, Zhejiang, China). Soluble Fms-Like Tyrosine Kinase-1 (sFlt-1) and placental growth factor (PIGF) was determined by quantitative sandwich enzyme immunoassays using commercial ELISA kits from Mybiosource San Diego, CA, USA.

Lactate dehydrogenase levels were measured in the media from cells subjected to EPS for 4 and 24h using the Lactate Dehydrogenase kit (Sigma). Cells were incubated in fresh media and the media collected at the appropriate time point. LDH release into the media was determined colorimetrically at 490nm according to the manufacturer’s instructions. Briefly, the lactate dehydrogenase assay mixture was prepared by mixing equal volumes of LDH assay substrate, dye and cofactor solutions. Twice the volume of LDH assay solution was added to the media sample and incubated in the dark for 30min at room temperature. The reaction was stopped by the addition of 1N HCl and the colour change read at 490nm. Total intracellular LDH for each cell culture was measured after the addition of 0.9% Triton X-100 and LDH release normalised to total LDH for each cell culture.