Cell proliferation assay kit

MCF10A and MDA-MB-231 cells were seeded into 96-well plates at a concentration of 2 × 10³ cells/well or 5 × 10³ cells/well, respectively. After 0, 24, 48, and 72 h, cell proliferation was assessed by a cell proliferation assay kit (BioVision) based on BrdU incorporation during DNA synthesis. Absorbance was measured using a plate reader (Epoch, Biotek) at 450 nm and expressed as arbitrary units.

LSCs were transfected with 10 nmol/L of siRNA targeting Gli1 or Gli3 genes (listed in Table 1; Stealth RNAi™ siRNA duplexes, Invitrogen) for 48 hours using the Lipofectamine RNAi MAX transfection kit according to the manufacturer's protocol (Invitrogen, USA). Scramble siRNA (sc‐37007, Santa Cruz Biotechnology) that did not specifically target any gene were used as control.
To evaluate the influence of Gli1 and Gli3 knockdown on LSC proliferation, LSCs (2 × 10³/well) were seeded in 96‐well cell culture plate (Costar; cat. no. 3599) one day before transfection. Forty‐eight hours after siRNA transfection, cells were maintained in serum‐free DMEM/F12 for 2 hours and then stimulated by 44‐mer (10 µmol/L) for 24 hours. The level of LSC proliferation was evaluated by Cell Proliferation Assay Kit based on the amounts of nuclear dye binding (BioVision; Catalog # K307‐1000), according to the company's instruction. All assays were performed in triplicate, and the experiment was independently performed for three times.

This study used the murine fibroblast cell line NIH-3T3 (AddexBio, San Diego, CA, USA) to assess the cellular response to recombinant Amwaprin. NIH-3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS (Gibco BRL) at 37 °C in a humidified incubator containing 5% CO₂. The cells were counted using a hemocytometer. The cell growth assessment of the NIH-3T3 cells was performed utilizing an MTT (3-(4,5 dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) Cell Proliferation Assay Kit (BioVision, Milpitas, CA, USA). NIH-3T3 cells (2 × 10⁴ cells/well of a 96-well plate) were incubated with serial dilutions of recombinant Amwaprin (0, 50, 250, or 500 ng/mL) or 1% Triton X-100 (Sigma-Aldrich) as a positive control that inhibits cell proliferation. Cells without Amwaprin and/or Triton X-100 were considered the negative control. The treated cells were incubated for 24 or 48 h, followed by the addition of 50 μL of the MTT reagent (BioVision) to each well. Following a 4 h incubation, the media was removed, and the formazan crystals were dissolved in 150 μL of MTT solvent. Absorbance was measured at 590 nm using a microplate reader (Infinite F50 Model, Tecan, Grödig, Austria). Additionally, the number of NIH-3T3 cells with or without recombinant Amwaprin (250 ng/mL) was counted for five days.

HaCat cells (1 × 10³/well) were seeded in 96-well cell culture plates (Costar; cat. no. 3599) precoated with the Fn (10 μg/mL; 50 μL per well) and incubated in 10% FBS culture medium for one day. HaCat cells cultured in uncoated wells served as a control. Subsequently, cells were maintained in serum-free DMEM for 3 h and then switched to stimulation medium, consisting of DMEM supplemented with 2% FBS and 10 μM peptide, for a further 24 h. Cell proliferation was evaluated by the Cell Proliferation Assay Kit (BioVision; Catalog # K307-1000, Milpitas, CA, USA), following the instruction manual. All assays were performed in triplicate, and the experiment was repeated 4 times.

To examine whether ABT-751 suppressed cell proliferation, a Cell Proliferation Assay Kit (Fluorometric, #K307-1000, BioVision) was used. It is based on a nuclear dye that specifically binds to nucleic acid in the cell and generates green fluorescence. The generated fluorescent intensity is directly proportional to the cell number, which can be quantified by measuring fluorescence (excitation/emission: 480 nm/538 nm). Shortly, cells (5 × 10³) were seeded in a 96-well plate with 100 μL of culture medium overnight, transfection of pSKP2-HaloTag or pHRIG-AKT1 was performed for 18 h, treated with DMSO (control) or ABT-751 for another 24 h. Precisely 25 μL of 5 × Nuclear Dye/Cell Lysis Buffer was added into each well and gently shaken with a digital rotator at room temperature protected from light for 15 min and subjected to be analysis.

Leukocytes were co-cultured with allogeneic normoxic and hypoxic MSCs (10:1). The leukocyte proliferation was assessed after co-culture with MSCs by flow cytometric analysis (BD Accuri). Briefly, after 72 h of co-culture, the leukocytes in the supernatant were collected and centrifuged at 1000 r.p.m. for 5 min. The pellet was washed three times using PBS and suspended in 100 μl of cold PBS. After fixing with 5 ml of 70% ice-cold ethanol, the cells were treated with RNase (20 µg/ml) for 30 min. The leukocytes were then stained with propidium iodide (5 μg/ml) for 5 min at RT and analyzed using flow cytometry. To measure leukocyte proliferation, cell cycle analysis was done by counting the number of cells entering S-phase (proliferating phase) and G2/M phase from G0/G1 phase (resting cells) of the cell cycle. The leukocyte proliferation was also measured by a Cell Proliferation Assay Kit (Biovision Inc. Cat # K301).

MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium] Cell Proliferation Assay kit (Biovision Inc) was used according to the manufacturer’s instructions to determine the effects of different drugs on the cell viability of HL-1 cells and hCAVSMCs. HL-1 or hCAVSMC cells were seeded (5 x 10³cells/well) in 96 well plates in complete Claycomb medium [38 (link)] or Medium 231 supplemented with SMGS, respectively and all incubations were performed at 37°C in the presence of 5% CO₂. After confirming that the cells were attached (as determined by light microscopy analysis), culture medium was removed and 200μL of serum-free Claycomb medium was added to HL-1 cells and 200μL of medium 231 without SMGS was added to CAVSMCs. Cells were then subjected to drug treatments by following a time course identical to those used for the xCELLigence RTCA. At the end of treatment, 20μL of MTS was mixed with culture medium and absorbance was measured at 30 min intervals at 490nm using the Synergy H4 Hybrid plate reader (BioTek, Vinooski, VT). Data is presented as the percentage of absorbance in drug treated cells compared to untreated cells.