Ldh kit

WS₂ powder, having an average nanoparticle size of 80 to 100 nm, was purchased from Lower Friction Company (Mississauga, ON, Canada). P(3HB-co-12 mol% HHx) powder (350,000 Da) was obtained from KANEKA Corporation, Osaka, Japan. Medium molecular weight chitosan powder was purchased from Sigma-Aldrich, St. Louis, MO, USA. Glacial acetic acid (99.8%) was obtained from Sigma-Aldrich, Petaling Jaya, Malaysia. Bacterial cultures of E. coli K1 and MRSA were grown and incubated for up to 16 h prior to antibacterial analysis. LDH kit was purchased from Roche Diagnostics, Indianapolis, IN, USA. Non-essential amino acid was purchased from Life Technologies, Carlsbad, CA, USA. In this experiment, analytical-grade reagents were used.

Adhering to the instructions of the lactate dehydrogenase (LDH) kit (Roche, NJ, United States, 04744926001) (an index of cytotoxicity), LDH secreted from neurons at 0, 24, 48 h prior to OGD was determined. The neurons were collected for analysis at 3 h following OGD. LDH activity = (absorbance value_{rm the sample}–absorbance value_{the control})/(absorbance value_{the standard}–absorbance value_{blank control}) × concentration_{the standard} (0.2 mmol/L) × 100%.

For cell death detection, LDH release was quantified using a cytotoxicity detection (LDH) kit (Roche #11644793001, Darmstadt, Germany) and was conducted following the manufacturer’s instructions. Absorbance was examined using an Infinite F200 pro Microplate Absorbance Reader at 490 nm.

Cytotoxicity was performed with the LDH kit (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer's instructions. Briefly, for effector cell preparation, Japanese flounder were injected i.m. with 20 µg poly(I:C) or PBS (control). At 5 d post-injection, PBL were prepared as described above and designated as effector cells. For target cell preparation, flounder were infected with megalocytivirus as described above. At 5 d post-infection (dpi), PBL were prepared as described above and designated as target cells. For LDH assay, the target PBL cells were distributed into a 96-well U-bottomed plate (1×10⁵ cells/well), and the effector PBL were added to the plate to the ratios (effector∶target) of 1∶1, 2∶1, 4∶1, and 8∶1. The plate was incubated at 22°C for 24 h and centrifuged at 250 g for 10 min. Aliquots (100 µl/well) were transferred to a fresh 96-well flat-bottom plate. To determine the LDH activity in the culture supernatant, an equal volume of freshly prepared reaction mixture (from the LDH kit) was added to the plate. The plate was incubated in the dark at room temperature for 30 min, and absorbance at 492 nm was measured. Cytotoxicity was calculated using the following formula: cytotoxicity (%) = {[A₄₉₂ (effector/target)−A₄₉₂ (effector control)−A₄₉₂ (target control)]/[A₄₉₂ (target maximum)−A₄₉₂ (target control)]}×100%.

Cell membrane integrity was determined using a commercial lactate dehydrogenase (LDH) kit (Roche, Basel, Switzerland) according to the protocol company’s instructions. The decrease of cell membrane integrity (e.g., increase in LDH levels) indicates cytotoxicity. In brief, the LDH levels in cell culture medium were assessed after cells treated with isoflurane and/or mild hypothermia. The amount of product was measured spectrophotometrically at a wavelength of 490 nm (SLT, Spectra). The cell integrity index was calculated as followed: LDH levels in the supernatant/ the total LDH levels.