Ldh cytotoxicity detection kit

After each treatment, all supernatant media was collected to evaluate the lactate dehydrogenase (LDH) release from the cytoplasm of damaged RGCs. The assay was performed using an LDH cytotoxicity detection kit (Promega, Fitchburg, WI, USA) according to the manufacturer’s instructions. Briefly, 50 μl of reconstituted substrate mix (Promega LDH kit) was added to each sample; after incubation at 25°C in the dark for 30 min, the enzymatic reaction was stopped with 50 μL of stop solution (Promega LDH kit). Absorbance was measured at 490 nm using a microplate reader (Synergy H1, BIOtAK). All experiments were performed in triplicate.

Cell lines were cultivated in the absence or presence of different concentrations of AR-12 at 37 °C and 5% CO₂ for 24 h, and then the culture supernatants were collected. Cytotoxicity of the AR-12-treated cells was determined using the lactate dehydrogenase (LDH) assay and detected by a microplate reader (Life Technologies). The levels of LDH released into the cell culture supernatants were measured using the LDH Cytotoxicity Detection Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The cytotoxicity percentage was calculated as follows: cytotoxicity % = (experimental group − medium background)/(Triton X − 100 − treated group − medium background) × 100%. The results of the three experiments are represented and expressed as the mean ± standard deviation.

Cell viability was measured as described previously with slight modification using the LDH cytotoxicity detection kit (Promega, Madison, WI), which quantifies the release of LDH from cells into the culture medium (Yoon et al., 2010[23 (link)]). Macrophage cells (1.5 × 10⁵ cells/mL) were seeded in 96 well plates, pre-incubated for 18 h, and then treated with LPS (1 μg/mL) plus IKCA (12.5 to 100 μM) at 37 °C for 24 h. Supernatants from the cultures were collected and used in the LDH assay, as instructed by the manufacturer. The optical density of the solution at a wavelength of 490 nm was measured using a microplate reader (Power Wave, Bio-Tek Inc., Winooski, VT). Percent cytotoxicity was determined relative to the control group. All experiments were performed in triplicate.

RAW264.7 cells, pretreated with different concentrations of inhibitors, shLuc-RAW264.7 cells or shGSK-3β-RAW264.7 cells were infected with GAS or its isogenic mutants at MOI of 25 as previously described. After cultivation for an additional 18 h with DMEM containing 50 μg/ml of gentamicin, the culture supernatants of the GAS-infected cells and uninfected control group were collected. The levels of lactate dehydrogenase (LDH) released into the cell culture supernatants were measured using the LDH Cytotoxicity Detection Kit (Promega) according to the manufacturer’s instructions, and detected by microplate reader (Life Technologies). The LDH release % = (experimental group-medium background)/(Triton X-100-treated group - medium background) × 100%. The results of three experiments are represented and expressed as the mean ± standard deviation.

DU145 and LNCaP cells were maintained at low passages (below 30) and cultured in RPMI 1640 (Wisent Bioproducts, QC) with 5 and 10% fetal bovine serum, respectively. For MTT proliferation assays 3500 LNCaP (on poly-L-lysine coated wells) or 1500 DU145 cells were seeded in 96-well plates, and after 24 h, the cells were treated with various concentrations of inhibitors (from 300 to 1 μM). Next, the cells were incubated for 72 h with peptides prior to addition of MTT reagent (Sigma–Aldrich, Canada) at a final concentration of 1 mg/mL. Formazan salt was solubilized with 50 μL DMSO and the metabolic activity was normalized relatively to vehicle-treated cells (sterile bi-distilled water). IC₅₀ values were determined using Prism 5.0 (GraphPad Software, USA), as previously described^{12 (link)}. For LDH assay, the LDH Cytotoxicity Detection Kit (Promega, USA) was used to quantify the release of lactate dehydrogenase following membrane disruption. As described in our previous work^{15 (link)}, DU145 cells were seeded at a density of 3000 cells/well and allowed to incubate for 24 h. Then, the media was subsequently replaced and cells were incubated with compounds at the selected concentration. Cells treated with 1% SDS were used as a control for maximal LDH release and further relative quantification.