Ldh cytotoxicity assay kit 2

Here, 10 ul of supernatant from each well was collected at 24, 48, and 72 h in culture. Then, LDH (LDH Cytotoxicity Assay Kit II, BioVision, Milpitas, CA, USA) reaction mix solution was added to the corresponding well of the 96-well plate and incubated in the dark for 30 min at room temperature. The OD values were measured at 450 and 650 nm (reference).

An LDH-Cytotoxicity Assay Kit II (BioVision, Milpitas, CA, USA) was employed according to the instructions of the manufacturer. Accordingly, 10 µL of the extracted cell supernatants were mixed with 100 µL LDH reaction reagent at room temperature for a time interval of 30 min. Thereafter, stop solution was added and absorbances were determined by using a multi-well spectrophotometer (ELISA reader) with filters for 450 nm and 650 nm (reference wavelength).

SH-SY5Y cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, PAA Laboratories) supplemented with 10% heat-inactivated fetal bovine serum and 2% Pen/Strep (PAA Laboratories) at 37°C with 10% CO₂. Cells were seeded into 96-well plates at a density of 50 000 cells/well in 100 μl cell culture medium and grown at 37°C. After 24 h the medium was removed and fresh medium was added together with the Aβ samples or controls to be analyzed.
The Aβ samples were obtained by initially dissolving disaggregated Aβ at high concentration in 100% DMSO. The peptide was then quantified and diluted to a final concentration of 100 μM into 50 mM HEPES buffer pH 7.4, containing 50 mM NaCl. If applicable, the solution also contained 20 μM B10 or KW1, and it was incubated for different periods of time at 37°C. After incubation the Aβ-containing analytes were added to the cells to reach a final concentration of 1 μM Aβ peptide. The cells were then incubated for 24 h at 37°C with the analytes before measuring the cellular effects with one of two assays.
Cell metabolic activity was assessed with a FLUOstar Omega 96-well plate reader (BMG LABTECH) by using the Cell Proliferation Kit I (MTT, Roche) or LDH-Cytotoxicity Assay Kit II (BioVision) according to the manufacturer’s protocol. Statistical analyses were carried out using the paired t-test implemented with SigmaPlot11 (Systat software).

The cytotoxic effects of GAS strains on A549 cells were determined by measuring the levels of lactate dehydrogenase (LDH) released from GAS-infected cells. A549 cells were infected by GAS at different MOIs (5, 10, or 20) and then incubated for 4 h. The supernatants of the infected cell cultures were collected and subjected to the assay. The level of LDH was measured using LDH-Cytotoxicity Assay Kit-II (K313-500, BioVision), according to the manufacturer’s instruction. The LDH levels of uninfected cells lysed with 1% Triton X-100 were served as 100% cell death with maximal LDH release.