Cell proliferation capacity was analyzed by Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) as described previously (28 (link)). Briefly, the cells were seeded into 96-well plates (Corning Incorporated, Corning, NY, USA) at a density of 103 cells/well and incubated for 1–7 days under the conditions stated previously. In total, 10 µl CCK-8 was added to each well, according to manufacturer's protocol, followed by incubation at 37°C with 5% humidified CO2 for 2 h. The absorbance of the cells was measured at 450 nm with Thermo Multiskan™ Spectrum microplate reader (Thermo Fisher Scientific, Inc. CCK-8 allows sensitive colorimetric assays for the determination of cell proliferation by measuring dehydrogenases in viable cells. All assays were carried out independently three times.
Thermo multiskan spectrum microplate reader
Manufactured by Thermo Fisher Scientific
The Thermo Multiskan™ Spectrum microplate reader is a versatile instrument designed for absorbance measurements in microplates. It provides accurate and reliable data for a wide range of applications, including cell-based assays, enzyme-linked immunosorbent assays (ELISAs), and quantitative analyses.
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2 protocols using thermo multiskan spectrum microplate reader
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Cell Proliferation Assay via CCK-8
2
Measuring Interleukin-6 Levels in Serum
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Serum samples were analysed for interleukin-6 concentration using commercially available human interleukin-6 enzyme linked immunosorbent assay (ELISA) kits (Quantikine ® , R&D systems), according to the manufacturer's instructions. Briefly, the serum samples were thawed and 200 μl aliquots of each sample, positive control or standard, were plated on a coated (monoclonal antibody specific for human interleukin-6) 96-well microtitre plate for 2 h. After washing, human interleukin-6 conjugate (200 μl) was added to each well and incubated for 2 h at room temperature (between 22°C and 24°C), with mixing on an orbital shaker. After the wells were washed, substrate solution (200 μl) was added to each well at room temperature (between 22°C and 24°C) and protected from light.
After adding 50 μl stop solution to each well, plates were measured with a Thermo Multiskan Spectrum microplate reader (Thermo Fisher Scientific) at 450 nm and values were calculated with Excel 365 (Microsoft, v. 365 ) by generating a four-parameter logistic (4-PL) curve fit. The minimum detectable dose of human interleukin-6 was 0.70 pg/ml.
After adding 50 μl stop solution to each well, plates were measured with a Thermo Multiskan Spectrum microplate reader (Thermo Fisher Scientific) at 450 nm and values were calculated with Excel 365 (Microsoft, v. 365 ) by generating a four-parameter logistic (4-PL) curve fit. The minimum detectable dose of human interleukin-6 was 0.70 pg/ml.
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