Cytotoxicity detection kit

The cell cytotoxicity assay was performed using the Cytotoxicity Detection Kit (Sigma Aldrich, St. Louis, MO) according to the manufacturer’s protocol. Briefly, 3 × 103 cells were seeded per well into 96-well plates and incubated overnight. After exposure to CWP232291, 50 μL of sample medium (from control and concentration dependent treatments) were transferred to a 96-well plate in triplicate wells. Reaction mixture (50 μL) was then added to each sample and incubated for 30 min in the dark at room temperature. The reaction was stopped by adding 50 μL of stop solution and mixing by gentle tapping. The absorbance was measured at 492 nm and 680 nm. The 680n m absorbance value (background) was subtracted from the 492 nm absorbance values before calculation of % cytotoxicity. The maximum lactate dehydrogenase (LDH) activity was determined after treating cell with lysis buffer as described in the manufacturer’s protocol. Cytotoxicity (%) was calculated using the following formula:
Cytotoxicity (%) = (CWP-treated LDH activity-Low LDH activity) / (High LDH activity-Low LDH activity) X 100.

LDH release was measured by colorimetric method using Cytotoxicity Detection Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. The concentration of released LDH from 100% cell lysis (OLs lysed with 1% Triton X-100) was used as the positive control and supernatants from OLs supplemented with 10% PBS as negative control (spontaneous LDH release). The rate of lysed cells was calculated based on the normalization of each sample to the level of LDH released by positive control subtracted from negative control samples. All samples were analyzed in duplicate.

Primary Human Aortic Endothelial cells (HAoECs) were obtained from PromoCell GmbH, Heidelberg, Germany. The cells were cultured in Endothelial Cell Growth Medium MV2 (PromoCell), passaged by treatment with Trypsin/EDTA (0.04%/0.03%) and grown in 48-well plates (Thermo Scientific, Roskilde, Denmark) coated with 1% gelatin (Sigma, St Louis, MO). In the experiments the cells were cultured with and without recombinant human (rh)IL-27 (100 ng/mL; R&D Systems, Minneapolis, MN) in Opti-MEM reduced serum medium (Gibco, Grand Island, NY) supplemented with 5% FBS for 1 h before stimulated with different concentrations of chemically synthesized hemozoin (Invivogen, San Diego, CA) for 22 h. For evaluation of possible cell toxicity different concentration of hemozoin was tested in HAoEC cultures using Cytotoxicity Detection Kit from Sigma Aldrich (St. Louis, MO).

The cells were plated in 96-well plates (3–5 × 10³/well) for 12 h and were treated as described before. LDH in the medium was then detected using the Cytotoxicity Detection Kit (Sigma-Aldrich, St. Louis, MO, USA). LDH assays were carried out according to the manufacturer’s instructions. The LDH level in cells exposed to Triton X-100 was considered as 100% to normalize the results. The absorbance was measured at 490 nm using the Multiskan™ FC Microplate Photometer.

Cell viability after treatment was measured by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay (M5655, Sigma-Aldrich) by adding 0.5 mg/mL MTT for 3 h in an incubator at 37°C and 5% CO₂. The cells were lysed using 0.1 M isopropanol/HCl +2% Triton-X100 for 1 h under gentle shaking at room temperature. Absorbance was finally read at 570 nm using a microplate reader (BMG Labtech, POLARstar Omega). Cytotoxicity was evaluated by measuring the activity of the lactate dehydrogenase (LDH) released in the supernatants of cells after the last 24 h of treatment according to the manufacturer’s instructions from the cytotoxicity detection kit (11644793001, Sigma-Aldrich).