Cellquanti blue cell viability assay kit

Cell proliferation was determined using Cell Count Kit-8 (CCK-8, Cat: 40203ES60, YEASEN, Shanghai, China). Cell viability was determined using CellQuanti-Blue^™ Cell Viability Assay Kit (CQBL-05 K, BioAssay systems). About 5000 cells (including RIPK1-knockdown cells GM12878/L428 and RIPK1 overexpression cells TMD8/U2932) were seeded in a 96-well microplate containing a culture growth medium for 12 h. Then, cells were subjected to a cell proliferation/viability assay every 12 h (0, 12, 24, 36, and 48 h) following the manufacturer’s instruction. Each sample was detected with at least three replications.

L929 mouse fibroblasts (CCL-1, ATCC) were cultured in Dulbeccoʼs Modified Eagle Medium (DMEM, PAN BIOTECH, Aidenbach, Germany) with 4.5 mg Glucose, 10% fetal calf serum (FCS), 1% Penicillin/Streptomycin and 3.7 g/L NaHCO₃. 1x10⁴ L929 mouse fibroblasts were seeded directly on the hydrogel specimen with 200 μL culture medium per well and incubated under cell culture conditions (37°C, 5% CO₂) for 46 hours (fibroblast cell density 62500 cellsxcm²). To assess relative cell viability, a resazurin based CellQuanti-Blue Cell Viability Assay Kit (BioAssay systems, Hayward, CA, USA) was implemented. 10% CellQuanti-Blue supplement was added to the wells followed by an incubation of another 2 hours under same conditions. The metabolic turnover from resazurin to the fluorescent resorufin (absorption 544 nm, emission 590 nm) was detected with the Fluostar optima (BMG LABTECH, Ortenberg, Germany). Replicates (n ≥ 3) were tested for normal distribution and subjected to a Nalimovs’ test for outliers. Means and standard errors (SEM) were calculated in Sigma Plot.

Camptothecin and SN-38 were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Ko143 was purchased from Sigma Aldrich Co. LLC (St. Louis, MO, USA). Verapamil and rifampicin were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Baicalin was purchased from AdooQ Bioscience (Irvine, CA, USA), and Cell Quanti-Blue^TM Cell Viability Assay Kit was purchased from Bio Assay Systems (Hayward, CA, USA). All other reagents were of high-purity analytical or high-performance liquid chromatography (HPLC)-grade. Pooled human serum from healthy subjects (normal serum) was purchased from Merck Millipore Ltd. (Billerica, MA, USA), and pooled human serum from patients with ESKD (uremic serum) was obtained from more than 400 patients with ESKD undergoing hemodialysis at Shirasagi Hospital (Osaka, Japan). Blood samples were collected immediately before hemodialysis in a non-invasive manner. Normal and uremic serum were deproteinized by 3-times volume of methanol as much as serum and evaporated under a nitrogen stream at 50 °C; the resulting serum residues, normal serum residue (NSR) and uremic serum residue (USR), were used in the study^{3 (link)}. The present study was approved by the ethics committees of Shirasagi Hospital (IRB number J2012013) and Kyoto Pharmaceutical University (IRB number 08-04).

Sodium butyrate (Sigma-Aldrich, St. Louis, MO) was dissolved in DMEM/F12 to make the butyrate working solution. Cells were treated with butyrate throughout the differentiation period (day 0 to 7). On day 7 post-differentiation, fresh medium was added and cells were challenged with LPS-derived from E. coli O55:B5 (Sigma-Aldrich, St. Louis, MO) at 10 μg/ml. Preliminary experiments were also conducted to determine the optimal butyrate concentration and duration of exposure to LPS to determine effect on cell viability. Analysis of cell viability was performed in 96-well cell culture plates (Corning Inc., Corning, NY) with the use of CellQuanti-Blue^TM cell viability assay kit (BioAssay Systems, Hayward, CA), according to the manufacturer’s instruction. The results of pilot experiments indicated that optimal concentrations of butyrate ranged from 0.1 mM to 1 mM because there was no significant adverse effect on cell viability at these concentrations (S1 Fig). Stimulation with LPS at 10 μg/ml LPS did not cause any cytotoxic effects up to 24 h of culture. There were 4 to 6 replicates per experiment.