Ldh cytotoxicity detection kit

After cell death, lactate dehydrogenase (LDH) was released into the supernatant, we used the LDH cytotoxicity detection kit (Beyotime Institute of Biotechnology, China) to monitor LDH released by H9C2 cells into the medium to determine cytotoxicity. LDH release reagent treatment (1:10 dilution, 1 h) was used as a positive control to test maximum LDH release. The optical density was measured spectrophotometrically at 490 nm on a microplate reader [19 (link)].

CHO cells were cultured in RPMI medium (Thermo Fisher Scientific, Shanghai, China) supplemented 10% FBS (Thermo Fisher Scientific, Shanghai, China), respectively at 5% CO₂ and 37°C. Exponentially growing P. aeruginosa bacteria cultured in casamino acids medium (OD₆₀₀ ≈ 0.5) were diluted with cell culture medium (OD₆₀₀ ≈ 0.1) and added to near-confluent CHO cells at a starting multiplicity of infection (MOI) ratio of 5: 1. After incubation at 37°C for 6 h, the extent of cell killing was determined by quantification of the release of lactate dehydrogenase into the cell culture supernatant using the LDH Cytotoxicity Detection Kit (Beyotime, Nantong, China).

The release levels of LDH in the culture medium were measured using the LDH cytotoxicity detection kit (#C0017; Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer’s instructions [49 (link)]. In brief, one group of wells was selected as a “maximal LDH release rate group.” Before treating cells as per the method in the “Cell culture and stimulation” section, cells were washed with PBS and the medium was replaced with a serum-free medium. LDH release reagent was added to the wells in the ‘maximal LDH release rate group’ 1 h before the end of treatment. At the end of treatment, the plate was centrifuged at 400× g for 5 min at room temperature. A total of 120 μL supernatant was transferred to a new plate. Subsequently, a 2-p-iodophenyl-3-nitrophenyl tetrazolium chloride and lactic acid solution was added to each well and the plate was incubated in the dark at room temperature for 30 min. The OD at 490 nm was measured and the release rate of LDH was calculated according to the following equation:
Release rate of LDH = (OD of treated sample-OD of control)/(OD of ‘maximal LDH release rate group’-OD of control) × 100%

DSPC, cholesterol and CP were purchased from Sigma Aldrich. Alum adjuvant was purchased from Thermo Fisher. Human Mincle-Fc and hMincle-his proteins were purchased from SinoBiological and Novoprotein, respectively. MCF-7, CT-26 and B16-F10 cancer cells were purchased from American Type Culture Collection (ATCC). Minimum Eagle's medium (MEM), RPMI medium 1640 and fetal bovine serum (FBS) were purchased from Gibico. Trypsin–EDTA was purchased from Invitrogen. HRP-linked goat anti-mouse kappa, IgM, IgG1, IgG2a, IgG2b, and IgG3 antibodies were purchased from Abcam. The FITC-labeled goat anti-mouse IgG antibody and LDH Cytotoxicity Detection Kit were purchased from Beyotime Biotechnology. Rabbit complements were purchased from Merck. Female BALB/c mice used for immunological studies were purchased from Southern Medical University (Guangzhou, China).

CAL27 and SCC25 cells were seeded into 96-well plates and cultured for 24 hours, and then they were treated with 125/100 μM oxaliplatin respectively. At four time points (0, 12, 24, and 48 hours), the LDH cytotoxicity detection kit (Beyotime, Shanghai, China) was used to detect cell mortality. The absorbance at 490 nm was measured, and cell mortality was calculated with the following formula: mortality (%) = (absorbance of processed sample - absorbance of sample control hole) / (absorbance of cell maximum enzyme activity - absorbance of sample control hole) × 100.