Wst 1 cell proliferation assay kit

The effect of different concentrations of NVP on cell viability/toxicity was analyzed using a nonradioactive WST-1 cell proliferation assay kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s instructions.

Cell proliferation was determined either by direct cell counting or using the WST‐1 Cell Proliferation Assay Kit (Roche Diagnostics GmbH, Mannheim, Germany).
For direct counting, cells were seeded at a density of 2 × 10⁴ cells (48‐well plates) and incubated at 37 °C for 24, 48, 72, or 96 h. Cells were detached from triplicate wells and counted in a hemacytometer. Cell death was evaluated by staining of the cells with trypan blue. At least three independent experiments were performed for each experimental condition.
For the WST‐1 assay, cells were seeded at a density of 5 × 10³ cells (96‐well plates) and incubated at 37 °C for 24, 48, 72, or 96 h. WST‐1 was added to each well and the plate was incubated in 5% CO₂ at 37 °C for 2 h. Plates were finally analyzed by measuring the absorbance at 450 nm versus the reference wavelength of 630 nm using a scanning multiwell spectrophotometer. Three independent experiments were performed for each experimental condition.

The cell proliferation assay was carried out using WST-1 Cell Proliferation Assay Kit (Roche). Forty-eight hours post-transfected cells or KO cells were seeded into 96-well plates at a density of 1000 cells/well. WST-1 reagents were added to each well at time point 0 h, 24 h, and 48 h, and cells were further incubated at 37 °C for 1 h. The absorbance was measured on a microplate Reader Infinite® M1000 PRO (TECAN) with a test wavelength at 450 nm and a reference wavelength at 630 nm The relative numbers of viable cells were estimated by subtracting the 630 nm background absorbance from 450 nm measurements.

Antibodies against STAT3, phosphorylated STAT3 (p-STAT3), C-Rel, and p-C-Rel were purchased from Bioss (Woburn, MA). WST-1 cell proliferation assay kit was obtained from Roche Diagnostics (Mannheim, Germany). FITC Anti-mouse B7-1 (Clone 16-10A1), PE Anti-mouse B7-2 (Clone GL1), PE Anti-Mouse IL-12 p40/p70 (Clone C15.6) and APC Anti-mouse CD11c (Clone HL3) antibodies were purchased from BD Pharmingen (San Diego, CA). IL-6 and IL-12 ELISA kit were obtained from R&D System Inc. (Minneapolis, MN). Anti-IL-6 (ab6672) and anti-CD11c (ab33484) antibodies for immunofluorescent (IF) staining were purchased from abcam (San Francisco, CA). Anti-IL-12 (NB600-1443) antibody for IF staining was purchased from Novusbio (Littleton, CO). Mouse sIL-6R alpha Simple Step ELISA Kit (ab203360) was obtained from abcam (Cambridge, MA). Cell Fixation/Permeabilization Kits (BD554715) for intracellular cytokine analysis was purchased from BD Bioscience (San Jose, CA). Purified Rat Anti-mouse IL-6 antibody (clone MP5-20F3) was obtained from BD Biosciences. STAT3 inhibitor VI, S31-201 was purchased from Santa Cruz Biotechnology.

Cell proliferation was measured using the WST-1 Cell Proliferation Assay Kit (Roche (Atlanta, GA, USA)), following the manufacturer’s instructions. H508, HT-29, and HCT116 cells were cultured at 50% confluence on 96-well flat-bottom plates. After serum starvation overnight, cells were treated with test chemicals for five days with or without 30-min pretreatment with inhibitors. Plates were read on a microplate reader at 440 nm 30 min after adding the WST-1 reagent. Cell proliferation rates were calculated according to the manufacturer’s protocol.