Celltiter blue reagent

Human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics (Lonza; Walkersville, MD, USA) and cultured according to the supplier’s instructions. Briefly, HUVECs from three to six passages were subcultured to confluence onto 0.2% gelatin-coated tissue culture flasks in endothelial cell growth medium (EGM2) containing growth factors and 2% FCS. All cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. Cell viability was assessed using Promega (Promega France, Charbonnière-les-Bains, France) CellTiter-BlueTM reagent according to the manufacturer’s instructions. Cells were seeded in 96-well plates (5 × 103 cells per well) containing 50 mL growth medium. After 24 h of culture, the cells were supplemented with 50 mL of test compound dissolved in DMSO (<0.1% in each preparation). After incubation for 72 h, 20 mL resazurin was added for 2 h before recording fluorescence (λex = 560 nm, λem = 590 nm) using a Victor microtiter plate fluorimeter (PerkinElmer France, Villebon-sur-Yvette, France). IC₅₀ values correspond to the concentration of test compound that elicited a 50% decrease in fluorescence for drug-treated cells relative to untreated cells. Experiments were performed in triplicate. Fumagillin was used as a positive control and showed an IC₅₀ of 0.006 ± 0.002 μM.

RBMCs were seeded at an initial density of 4 × 10⁴ cells/cm² onto the titanium surfaces of the three groups and allowed to attach to the surface for 1, 3, 6, and 24 h. A total of 50 μL CellTiter-Blue^® Reagent (Promega Corporation, Madison, WI, USA) diluted in 250 μL phosphate-buffered saline (PBS) was used to count the number of RBMC adhesions. The analysis was carried out in accordance with the manufacturer’s instructions. The cells were labeled and observed after 24 h of culture, as described in our prior research [30 (link),31 (link),32 (link),33 (link),34 (link)].

Human fetal lung fibroblasts MRC5 transformed with SV-40 (MRC5 SV2, EcACC Cat. No. 84100401) and primary skin fibroblasts from Common Use Center “Biobank” (Research Centre for Medical Genetics, Moscow, Russia) were cultured in DMEM (Dulbecco’s modified Eagle’s) medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 2 mM glutamine and 10% fetal bovine serum (FBS) (HyClone, Logan, UT 84321 USA) and 100 U/mL streptomycin and 100 U/mL penicillin (all from Gibco, USA). Cell viability was measured using the CellTiterBlue^® reagent (Promega, USA) according to the manufacturer’s protocol with Fluoroskan Ascent FL Microplate Reader (Thermo Labsystems, Waltham, MA, USA).

SHEP WT and SHEP T58/S62 were seeded in 384-well plates for 48 hours before treatment with 40, 200 and 1000nM of 228 kinase inhibitors and chemical probes for 96 hours [82 (link), 97 (link)–99 (link)]. Cell viability was determined using CellTiter-blue reagent (Promega, Inc.). The Z-factor was >0.5 for all plates analyzed. The data was plotted as a ratio of SHEP T58/S62:SHEP WT.

Cell viability was carried out by plating 1000 cells/well in 96-well plates using CellTiter-Blue viability assay according to the manufacturer’s protocol. Cells were treated as indicated for 72 h. CellTiter-Blue reagent (G8081, Promega) was added after 72 h treatment and cells were incubated for 1–2 h before recording fluorescence intensities (ex. 560/20 nm; em. 590/10 nm) using a Perkin Elmer Wallac 1420 Victor2 Microplate Reader. Trypan blue exclusion and cell viability measurements were performed using an EVE Automated Cell Counter (NanoEnTek, #E1000) and EVE Cell Counting Slides. 10 μL of cell suspension were mixed with 10 μL of 0.4% Trypan solution and a final volume of 10 μL of this mixture used to count cells and assess viability according to the manufacturer’s protocol.