Lactate dehydrogenase activity assay kit

Whole blood was collected via cardiac puncture and serum was obtained after centrifugation at 10,000 × g for 5 min at 4 °C. ELISA for IFN-γ (#88-7314-86), IL-6 (#88-7064-88), IL-1β (#88-7013-88), and TNF-α (#88-7324-88) (all from ThermoFisher Scientific) was performed according to the manufacturer’s protocol. Shortly, 96-well plates were coated overnight at 4 °C with the corresponding capture antibody. Then, they were washed and blocked with assay diluent for one hour. After washing, both samples and standard dilutions were incubated for 2 h at room temperature. Then, samples were washed and incubated with detection antibody and Avidin-HRP in assay diluent for 1 h at RT. Substrate solution was added until an appropriate blue color was produced. The reaction was stopped by adding 1 M H₂SO₄. The absorbance was measured using an absorbance reader. Serum samples were diluted 1:3 in dilution buffer. The measurement of myoglobin concentration in the serum was performed with the Mouse Myoglobin ELISA Kit (Abcam, #ab210965). The activity assays for the determination of the creatine kinase and lactate dehydrogenase were performed using the Creatine Kinase Activity Assay Kit (Abcam, #ab155901) and Lactate Dehydrogenase Activity Assay Kit (Abcam, #ab282925).

Chia seeds (Salba grain organic) were obtained from I.P.A. s.r.l. Industria Prodotti Agroalimentari (Viterbo, Italy). L-ascorbic acid, β-glycerophosphate, dexamethasone, Phosphate Buffer Saline (PBS), Tris-HCl, Triton X-100, magnesium chloride (MgCl₂), para-nitrophenylsphoshate, Bradford reagent and Alizarin Red S were all from Sigma. Lactate Dehydrogenase Activity Assay Kit was from Abcam. DMEM (Dulbecco’s Modified Eagle’s Medium) High Glucose, Fetal Bovine Serum (FBS), streptomycin, penicillin and trypsin were bought from EuroClone (Milan, Italy).

The LDH activity of cell lysates was examined using Lactate Dehydrogenase Activity Assay Kit according to the manufacturer’s instructions (BioVision, CA, USA). In brief, cells were collected, washed and extracted to measure LDH activity. The results were normalized to the amount of total protein.