Cell counting kit 8 (cck8)

Cepharanthine was purchased from Shiji-Aoke (Beijing, China), cell counting kit-8 was bought from APExBio (Houston, TX, United States), DMEM, penicillin, streptomycin, fetal bovine serum (FBS) and PBS were obtained from Gibco Life Technologies (Grand Island, NY, United States). Anti-GAPDH, P62 and anti-rabbit IgG HRP-linked antibody were obtained from Cell Signaling Technology, Inc., (Beverly, MA, United States). Cathepsin B antibody was purchased from the Santa Cruz Biotechnology (Santa Cruz, CA, United States). Primary antibodies against LC3 were obtained from Abcam (Cambridge, MA, United States), anti-rabbit HRP-conjugated secondary antibody and anti-mouse HRP-conjugated secondary antibody were purchased from Cell Signaling Technology (Boston, MA, United States).

Cell proliferation assays were performed using a Cell Counting Kit-8 (CCK-8, APExBio, USA). Proliferation rates were determined 0, 24, 48, 72, and 96 h after infection, and the absorbance was measured at 450 nm following the manufacturer-recommended protocol. The apoptosis rate was confirmed by flow cytometry (FCM) using an Annexin V-FITC/PI apoptosis detection kit (Elabscience Biotechnology Co., Ltd., Wuhan, China). Cell cycle analysis was performed using a Cell Cycle Analysis Kit (Sigma, St. Louis, MO, USA).

The proliferation of ECs with different treatments was assessed using Cell Counting Kit-8 (CCK8; K1018, APExBIO)). In brief, ECs were seeded at an initial density of 5 × 10³ cells/well in 96-well plates. After different treatments, 10 μL CCK8 solution was added into each well of the palate for 12, 24, 48, and 72 hours. Then, the palate was incubated for 3 hours. The optical density value was assessed by a microplate scanning reader (BioTek Instruments) at 450 nm.

Cell viability was determined by Cell Counting Kit-8 (K1080, Apexbio Technology LLC, Houston, TX, USA). CRC were seeded into 96-well plates or 24-well plates for 24 h. The cells were treated with indicated doses of DIM, 5-FU, and the combination of DIM and 5-FU for 48 h. Then, 10% (v/v) CCK-8 solution was prepared and added to every well, and cells were plated into a cell incubator for 3 h [42 (link)]. Optical density was measured at 450 nm using Epoch™ Multi-Volume Spectrophotometer and Take3™ (BioTek, Winooski, VT, USA). Cell viability was normalized as a percentage of the negative controls treated with DMSO.

Cell viability was determined by the Cell Counting Kit-8 assay (CCK-8, APExBIO, Houston, TX, USA) according to the manufacturer’s instructions. Briefly, TCMK-1 cells in logarithmic growth phase were seeded in 96-well culture plates at a density of 5000 cells/well. After treating with BMS309403 or LPS, a 10 μl CCK-8 solution was added to each well and incubated in dark for 1 h at 37°C. In the end, the absorbance at 450 nm was detected using a microplate reader (Synergy Mx, Biotek, Vermont, USA).

For growth curve, cell viability was monitored by the IncuCyte live cell analysis system (Essen BioScience, Ann Arbor, MI, USA). To assess the cell proliferation, CRC cells were equally seeded into fresh 12-well plates at a density of 1 × 10^{5 (link)} cells. Cells were treated with different indicated concentrations of C.B CM. After placing the cell in a cell incubator, the IncuCyte live cell analysis system analyze the photographs at 9 random fields to obtain growth curve.
For IC50 calculation, cell viability was determined by Cell Counting Kit-8 (K1080, Apexbio Technology LLC, Houston, TX, USA). CRC cells were seeded into 96-well plates for 24 h. The cells were treated with indicated doses of C.B CM, 5-FU, and the combination of C.B CM and 5-FU for 48–72 h. Following that, 10% (v/v) CCK-8 solution was prepared and added to every well, and cells were plated into a cell incubator for 24 h. Optical density was measured at 450 nm using Epoch™ Multi-Volume Spectrophotometer and Take3™ (BioTek, Winooski, VT, USA). Cell viability was normalized as a percentage of the negative controls treated with culture medium. IC50 values were calculated by using linear regression equations obtained from the concentration vs percentage of viable cells.

Cell viability was quantified using the Cell Counting Kit‐8 (K1018, ApexBio, USA) as per the instructions of the manufacturer. For colony formation, 10³ cells were harvest and put into 6‐well plates and allow to proliferate for 10 days. The obtained colonies were fixed using paraformaldehyde (4%) for 10 minutes and stained with crystal violet (1%) for 20 minutes.