The transfected cells were diluted into 96-well plates at a density of 5 × 103 cells per well. Each well was subjected to the Cell Counting Kit-8 (CCK8) assay (Solarbio, China). The cells were incubated for 1 h in the dark in a 37°C incubator. Absorbance was recorded at 450 nm with a microplate reader. Cell viability and proliferation rates were calculated after the detection values were obtained.
Cell counting kit 8 cck 8 assay
Manufactured by Solarbio
Sourced in China
The Cell Counting Kit-8 (CCK-8) assay is a colorimetric assay used for the determination of cell viability and cytotoxicity. It utilizes the tetrazolium salt WST-8 to produce a water-soluble formazan dye upon reduction by dehydrogenases in viable cells. The resulting color change can be measured spectrophotometrically, providing a quantitative assessment of cell proliferation and survival.
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7 protocols using cell counting kit 8 cck 8 assay
1
Cell Viability and Proliferation Assay
2
AngII-Stimulated Cell Proliferation Assay
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Cell proliferation was evaluated using a Cell Counting Kit-8 (CCK-8) assay (Beijing Solarbio Science & Technology Co., Ltd.). The NCFs were plated on 96-well plates at a density of 1×104. Following stimulation with AngII (200 nM) for 24 h, the NCFs were treated with or without different concentrations of VCP979 (0.1, 1, 3 and 9 µM) for another 24 h. According to the manufacturer's protocol, 10 µl CCK-8 solution was added to each well and the cells were incubated at 37°C for 1-2 h. A microplate spectrophotometer (M200pro) was used to read the optical density of each well at 450 nm.
3
Cell Colony Formation and Proliferation Assay
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To evaluate cell colony formation ability, transfected cells were plated into a 6-well plate (1x104 cells/well) and the cells were allowed to grow for 10 days at 37˚C. The colonies were observed after 3.7% paraformaldehyde fixation for 10 min at room temperature and 0.2% crystal violet staining (Shanghai Aladdin Biochemical Technology Co., Ltd.) for 5 min at room temperature.
A Cell Counting Kit-8 (CCK-8) assay (Beijing Solarbio Science & Technology Co., Ltd.) was used to assess cell proliferation. Briefly, transfected cells were incubated with CCK-8 solution for another 4 h at 37˚C before the reading of OD450 using a microplate reader (Bio-Rad Laboratories, Inc.).
A Cell Counting Kit-8 (CCK-8) assay (Beijing Solarbio Science & Technology Co., Ltd.) was used to assess cell proliferation. Briefly, transfected cells were incubated with CCK-8 solution for another 4 h at 37˚C before the reading of OD450 using a microplate reader (Bio-Rad Laboratories, Inc.).
4
Ginsenoside Rg3 Modulates Cell Viability
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Cell counting kit-8 (CCK-8) assay (Solarbio Science & Technology Co., Ltd., Beijing, China) was used to explore the effect of ginsenoside Rg3 on cell viability. In a 96-well plate, human peritoneal mesothelial cells (5×103 cells/well) were incubated for 24 h. Then, fresh medium containing different concentrations of ginsenoside Rg3 was prepared to replace the medium, and the cells were further incubated for 72 h. Cell viability was measured according to the manufacturer’s instructions. The final DMSO concentration was <0.1%. Treated cells were also labeled with 5-ethynyl-2′-deoxyuridine (EdU) using a Cell Light Edu Apollo 567 In Vitro Kit (Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer’s protocol. Images were captured using an Axio Imager M2 fluorescence microscope (Zeiss GmbH, Jena, Germany).
5
Assessing CAPE Biocompatibility on HOKs
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The biocompatibility of CAPE on human oral keratinocytes (HOKs) was detected by the Cell-Counting-Kit 8 (CCK-8) assay according to the manufacturer’s instructions (Beijing Solarbio Science & Technology Co., Ltd.). The HOK cell line was acquired from the State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, China. The cells were incubated in 100 U/mL penicillin supplemented with DMEM (Gibco) and 100 mg/mL streptomycin supplemented with 20% FBS (Gibco) in a volume of 100 μL per well in a 96-well cell plate with a final CAPE concentration of 20, 40, 80, or 160 μg/mL, the control group was prepared as described above. After incubation for 24 h, the cells were resuspended gently with sterile PBS, and 10 μL of CCK-8 reagent was mixed in every well. After another 4 h of cultivation, the absorbance at 450 nm was measured using a microplate spectrophotometer.
6
Neural Stem Cell Proliferation Assay
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To evaluate the effect of the scaffold on the proliferation of neural stem cells, the viability of neural stem cells is analyzed by Cell Counting Kit-8 (CCK-8) assay (Solarbio Science & Technology Co., Ltd); 100 μL of NSC was seeded on the 3D-C/S and 3D-C/S+ST in each well of the 96-well plate at a density of 1 × 109/L (five duplicate wells per group). At 1, 3, 5 and 7 days after co-culture, 10 μL CCK-8 solution was added to each well, followed by culture for 3 h. A multi-mode microplate reader (BioTek, Synergy2, USA) was implemented to detect the OD value at 450 nm.
7
Evaluating hUSLFs Response to 17β-Estradiol
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hUSLFs from POP patients and control at passage 3-7 were treated with different concentrations of 17 β-estradiol. The fibroblasts seeded at a density of 104 cells/well in 96-well plates and cultured overnight, then the culture medium was replaced by FBS-free DMEM and the cells were cultured for 6 h. The medium was then replaced by DMEM (supplemented with 10% FBS and 1% P/S) containing different concentrations of 17 β-estradiol (0, 10−11, 10−10, 10−9 and 10−8 M, MCE, United States)). Different concentrations of 17 β-estradiol were freshly prepared by dissolving the 17 β-estradiol powder in absolute dimethyl sulfoxide and then serially diluted with medium. Each group of the experiment was done in triplicates. After culturing for 24 h, the cell viability was measured by Cell Counting Kit-8 (CCK-8) assay (Solarbio, China) at 24, 48, 72 and 96 h according to the manufacturer’s protocol.
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