Cell viability was quantified using a cell counting kit-8 (CCK-8) assay (Promega, Kumamoto, Japan). Approximately 0.7 × 104 cells were seeded in each well of a 96-well plate containing 100 μL RPMI medium supplemented with 10% FBS, 100 μg/mL penicillin, and 0.25 μg/mL streptomycin and were cultured overnight. After 20 h of incubation, various concentrations of STK899704 were added to the wells and the cells were incubated further for the indicated time. Cell viability was analyzed by performing the CCK-8 assay according to the manufacturer’s instructions. Optical density was measured at 450 nm using a microplate reader (Apollo LB 9110; Berthold Technologies GmbH, Bad Wildbad, Germany). Also, cell viability was assessed by the MTS dye reduction assay, which measures mitochondrial respiratory function. Lung cancer cells were seeded (7 × 104cells/mL) in 100 μL medium/well in 96-well plates, incubated overnight, and treated with various concentrations of STK899704, as described in the figure legends. Cell viability was calculated by assessing MTS metabolism as previously reported (Kwon et al., 2016 (link)). In brief, media samples (100 μL) were removed and incubated with 100 μL of MTS-PMS mix solution for 1 h at 37°C. Optical absorbance was measured at 492 nm using an ELISA reader (Apollo LB 9110, Berthold Technologies GmbH).
Apollo lb 9110
Manufactured by Berthold Technologies
Sourced in Germany
The Apollo LB 9110 is a laboratory equipment product manufactured by Berthold Technologies. It is a versatile device designed for various analytical applications in research and testing environments.
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15 protocols using apollo lb 9110
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Cell Viability Assays of STK899704
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MTS Assay for Cell Viability
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Cell viability was examined by using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. The cells were seeded in 100 μL medium/well in 96-well plates (A549 cells: 0.7 × 104 cells/well; NCI-H1299 cells: 0.9 × 104 cells/well) and allowed to grow overnight. After 24 h, different concentrations of CG extract were added, and the cells were returned to the incubator for a further 24 or 48 h. Subsequently, the medium (100 μL) was removed and incubated with 100 μL MTS with PMS mix solution for 40 min to 1 h at 37 °C. The optical density at 492 nm was measured for each well by using an ELISA reader Apollo LB 9110 (Berthold Technologies GmbH, Zug, Switzerland).
3
Cell Proliferation Rate Estimation
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Cell proliferation rate was estimated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl) -2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay with a CellTiter 96 Aqueous One Solution Assay (Promega, USA). Primary and immortalized HDPCs (1.0 × 104 cells/well) were seeded overnight in 100 μl of the medium in 96-well plates and then incubated for 0, 24, 48, and 72 h. The reagent (20 μl) was added to each well and incubated for 30 min. The absorbance was measured at 492 nm using a microplate reader (Apollo LB 9110; Berthold Technologies GmbH, Bad Wildbad, Germany).
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MTS Assay for THP-1 Cell Viability
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Cell viability was estimated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. The CellTiter 96 Aqueous One Solution Assay (Promega, Madison, WI, USA) was used. THP-1 cells (3 × 104 cells/well) were seeded in 96-well plates in 100 μL of the medium. Moreover, the cells were co-treated with MMPP (2 and 4 µg/mL) and LPS (1 µg/mL), or MMPP (2 and 4 µg/mL) alone for 24 h. A 20 µL of the reagent was then added to each well, and the mixture was incubated for another 30 min. Absorbance at 492 nm was measured using a microplate reader (Apollo LB 9110; Berthold Technologies GmbH, Bad Wildbad, Germany).
5
Luteolin Cytotoxicity Evaluation
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MDA-MB-231 and HaCaT cells were seeded at approximately 1.5 × 104 cells per well in a 96-well plate and grown overnight. Then, the medium was replaced with medium containing various concentrations of luteolin and luteolin glycosides and the cells were cultured for another 24 h. The effect of luteolin and luteolin glycosides on cell viability was assessed using electron-coupling reagent containing MTS and PMS (99:1 ratio). These reagents were mixed with medium that was added to the plate at 100 μL per well, and the cells were incubated for an additional 1 h. The optical density at 492 nm was measured using a microplate reader (Apollo LB 9110; Berthold Technologies GmbH, Bad Wildbad, Germany). The percentage of viable cells was estimated relative to those in untreated controls. The cell viability assay was repeated thrice.
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MTS Assay for Cell Viability
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Cell viability was assessed using an MTS assay, according to the manufacturer’s instructions. Cells (1 × 103 cells/well) were seeded in 96-well plates and then treated with various concentrations of ML and TPA (50 nM) for 24 h. Absorbance was measured at 492 nm using a microplate reader (Apollo LB 9110; Berthold Technologies, Germany).
7
Cervical Cancer Cell Viability Assay
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Cell viability was assessed by the MTS dye reduction assay, which measures mitochondrial respiratory function. Cervical cancer cells were seeded (12 × 104 cells/mL) in 100 μL medium/well in 96-well plates, incubated overnight, and treated with various concentrations of CTS, as mentioned in the figure legends, for 24 h. Cell viability was calculated by assessing MTS metabolism as previously reported [10 (link)]. In brief, media samples (100 μL) were removed and incubated with 100 μL of MTS-PMS mix solution for 1 h at 37°C. Optical absorbance was measured at 492 nm using an ELISA reader (Apollo LB 9110, Berthold Technologies GmbH & Co. KG, Bad Wilbad, Germany).
8
Recombinant MD2 Protein Binding Assay
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The recombinant MD2 (rMD2, 1 μg/mL) (R&D Systems, Minneapolis, MN, USA) protein diluted in PBS was immobilized in 96-well plates (Thermo Fisher Scientific Inc., Waltham, MA, USA) and incubated at 37 °C for 2 h. The wells were washed with PBS, followed by blocking with 300 μL of 5% BSA in PBS at room temperature overnight. The plate was then washed with PBST (PBS with 0.05% Tween-20) [10 (link)]. MMPP and Dex were pre-incubated at 37 °C for 1 h. Then biotinylated LPS (100 ng/mL) (Invivogen, San Diego, CA, USA) was added and incubated at room temperature for 1 h. Streptavidin conjugated to horseradish peroxidase (HRP) was incubated at 37 °C for 1.5 h [37 (link)]. The activity of HRP was determined using KLP SureBlueTM TMB substrate, and the reaction was stopped by 2.5 N H2SO4. The optical density of each well was measured at a wavelength of 450 nm, using a microplate reader (Apollo LB 9110; Berthold Technologies GmbH, Bad Wildbad, Germany).
9
MTS Assay for 3T3-L1 Cell Viability
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Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. 3T3-L1 MBX cells were seeded in 100 μL complete culture medium in 96-well plates and treated with MMPP for 48 h. The effect of MMPP on cell viability was measured using the CellTiter 96 Aqueous One Solution Assay (Promega, Madison, WI, United States ) containing MTS and phenazine methosulfate, an electron-coupling reagent. Briefly, a 100-μL aliquot of the aqueous reagent solution was added to each well, and the cells were incubated for 1 h. Absorbance was measured at 492 nm using a microplate reader (Apollo LB 9110; Berthold Technologies GmbH, Bad Wildbad, Germany). The percentage of viable cells was estimated relative to that of the untreated controls.
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Evaluating Cell Viability with MTS Assay
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Cell viability was assessed by the MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxy methoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium) assay, which measures mitochondrial respiratory function. HT29 cells (3 × 104 cells/ml) were seeded into 96-well plates, incubated overnight, and treated with various concentrations of STK899704 for 24 h. Cell viability was calculated by assessing MTS metabolism, as previously reported (31 (link)). Briefly, media samples (100 μl) were removed and incubated with 100 μl of MTS-PMS (phenazine methosulfate) solution for 1 h at 37°C. The optical absorbance of the samples was measured at 492 nm using an enzyme-linked immunosorbent assay reader (Apollo LB 9110, Berthold Technologies GmbH &Co. KG, Bad Wildbad, Germany).
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