Cck 8 reagent

Transfected cells (2000 cells/pore) were seeded into 96-well plates for CCK-8 assay. Then, 10 μl of CCK-8 reagent (APExBIO, USA) and 100 μl of serum-free MEM medium were introduced into cells and incubated for 2 h. Subsequently, the absorbance was measured at 450 nm at 0, 24, 48, 72, and 96 h.

Cells were seeded into a 96-well plate at a density of 2 × 10³ cells/well, 5 duplicate wells (100 μl DMEM per well) were set up, then placed in an incubator at 37 °C with 5% CO₂. 10 μl of CCK-8 reagent (APExBIO, USA) was added in each well. After incubating at 37 °C for 1 h in the dark, the absorbance was detected at 450 nm wavelength with a microplate reader (Tecan, Switzerland) once a day for 4 consecutive days at the same time.

Cell counting kit-8 (CCK-8) was used to determine the survival rate by measuring the absorbance at 450 nm (A₄₅₀). Briefly, an equal number of cells were seeded into 96-well plates beforehand and then treated under different conditions. After that, cells were washed with PBS and a complete medium supplemented with 10% CCK-8 reagent (APExBIO, Boston, MA) was added to each well including the blank well (Blank control group). Then the plates were incubated in the cell incubator for about 2 h for further measuring A₄₅₀. The survival rate was calculated according to the formula survival rate = (A_{450 Treatment group} − A_{450 Blank control group})/(A_{450 Control group} − A_{450 Blank control group}). All the experiments were performed in triplicate and the data were analyzed by Student’s t-test or one-way ANOVA test.

The CCK-8 reagent was purchased from APExBIO (USA). The 2×10³ cells were seeded into 96-well plates in advance. The test was started 24 hours later and lasted for 6 consecutive days (0, 1, 2, 3, 4, 5, 6 days). The medium was absorbed and discarded, and CCK-8 reagent was mixed with serum-free medium in a 5ml eppendorf tube (CCK8 reagent: Serum-free medium =10 μL: 90 μL per well) and added into 96-well plates with 100 μL per well. OD value at 450 wavelength was detected on a microplate after incubation at 37°C for 1h.

CO₂ incubator was made in Japan from SANYO Electric Biomedical Co., Ltd. Multiskan Spectrum was purchased from American Thermo Fisher Science Co., Ltd. Inverted fluorescence microscope (ICX41) was purchased from Ningbo Shunyu instrument Co., Ltd.
CCK-8 reagent was purchased from APExBIO. Trypsin 0.25% solution, penicillin–streptomycin solution, and DMEM media were purchased from www.gelifesciences.com/hyclone. TRIzol Reagent (Total RNA Isolation Reagent) was purchased from American Thermo Fisher Science Co., Ltd. Fetal bovine serum (FBS) was purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co.,Ltd. 24-well, 6-well and 96-well culture plates were purchased from Wuxi NEST Biotechnology Co., Ltd. EP pipes (2, 5, 15 and 50 mL) were purchased from Wuxi NEST Biotechnology Co., Ltd.

For MTS cell growth assay, equal number of pancreatic cancer cells were plated into the 96 well plate and added MTS reagent according to the manufacture’s protocol (Cat No.Ab197010, Abcam). The absorbance at 490 nm was used for the evaluation of cell proliferation rate.
For CCK 8 assay, 1000 cells were placed in each well of the 96-well plate, and fresh medium containing 10ul CCK 8 reagent (Cat No. K1018, APExBIO) was replaced at the same time point of the first, second, third and fourth days, respectively, and incubated at 37° and 5% CO2 for 1 h. Absorbance value was measured at the wavelength of 450 nm.
For BrdU cell proliferation assay, 1000 cells were placed in each well of the 96-well plate. The adherent cells were washed with PBS three times, and the proliferation of the cells was detected with the BrdU cell proliferation kit according to the manufacturer’s instructions (Cat No. 6813, Cell Signaling Technology). Absorbance value was measured at the wavelength of 450 nm.