The effect of RTHF on A549 cell growth was examined using CCK-8 assay (7 Sea Biotech) following the manufacturer’s protocol. After differential exposure for 48 hours, 10 µL of WST-8 was added to each well and allowed to incubate for 4 hours at 37°C. Finally, the absorbance reading of each well was detected with microplate reader at 450 nm (Sigma 1-15K; Thermo Fisher Scientific). Decrease in the absorbance indicates increased cytotoxicity. The percentage of viable cells was estimated by comparison with untreated control cells.
Cck 8 assay
Manufactured by 7Sea Biotech
Sourced in China
The CCK-8 assay is a colorimetric cell proliferation and cytotoxicity assay. It measures the activity of cellular dehydrogenases, which reflect the number of viable cells in a sample. The assay is based on the reduction of a tetrazolium salt, WST-8, to a water-soluble formazan dye by cellular dehydrogenases. The resulting color change is proportional to the number of living cells and can be measured using a spectrophotometer.
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6 protocols using cck 8 assay
1
Cytotoxicity Assessment of RTHF
2
Cell Proliferation Modulation by PKCζ Inhibitor
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Cell proliferation was evaluated by CCK-8 assay (7Sea Biotech) according to the instructions provided by the manufacturer. Briefly, the cells were seeded in a 96-well plate at a density of 4.0×103 cells/well and incubated at 37°C for 24 h. The cells were then treated with or without PKCζ-specific pseudosubstrate inhibitor (10 µM; Invitrogen), After 3 h, the inhibitor mixture was removed and replaced with fresh medium alone or with TNFα (rhTNFα, 10 or 100 ng/ml; Shanghai Bioleaf Biotech Co., Ltd.) for 24, 48 and 72 h. At the indicated time point, the medium was removed and supplemented with the medium containing CCK-8 reagent (10 µl/well) and the cells were cultured for a further 1–1.5 h at 37°C, 5% CO2. Subsequently, the absorbance was measured at 450 nm using a Tecan Infinite M200 Pro Nano Quant micro-plate reader (Tecan).
3
Cell Proliferation Assessment by CCK8
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To measure the cell proliferation abilities, cells were transfected with indicated plasmids and then performed CCK8 assay (7 sea biotech, Shanghai, China).
4
Cell Viability Assay in 96-Well Plate
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Each well of a 96-well plate contained about 5,000 transfected cells. Cell viability was assessed using a cell counting kit-8 (CCK-8) assay (7 Sea Biotech, Shanghai, P.R. China). Following incubation for 24, 48, and 72 h, 10 μl of CCK-8 was added to each well and incubated for 2 h. The absorbance value was determined using a multimode microplate reader (Berthold Technologies GmbH & Co.KG, Bad Wildbad, Germany) at 450 nm.
5
VB1 Cytotoxicity Assay in Cultured Cells
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Cells were seeded into 96-well plates (5 × 103 cells per well) and cultured at 37°C in media containing 10% FBS for 24 h. Cells were exposed to VB1 (0 µM), 0.1% DMSO, or various concentrations of VB1 (2.5, 5, 10, and 20 µM) for 24, 48, and 72 h, respectively. The cell viability (%) was determined by CCK-8 assay (7 sea biotech, China) with the manufacturer’s instructions. In detail, the reagent (10 μl) was added into each well of the 96-well assay plate containing the samples in 100 ml of culture medium and incubated for another 4 hours, after 24, 48, and 72 h individually. The absorbance at a wavelength of 450 nm was measured using a microplate absorbance reader (Eppendorf, GER). The half-maximal inhibitory concentration (IC50) values were generated using the SPSS software.
6
Cell Proliferation Assay with Exosomes
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Cell proliferation was monitored using CCK-8 assay (7-Sea Biotech, Shanghai, China) following the manufacturer's protocol. Briefly, 2 × 10 3 cells per well were allowed to grow in 96-well plates, and were treated with exosomes that were derived from Nthy-ori-3-1 and BCPAP cells under normoxic or hypoxic conditions for Days 1, 2, 3 and 4. Ten microliters of CCK-8 reagent were added to each well at 4 h before the endpoint of incubation. The optical density (OD) 450 nm values were determined by a microplate reader (NanoDrop 2000) . All experiments were repeated at least three times.
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