The viability of cultured granulosa cells was quantified using an MTT assay. Briefly, cells were seeded in 96-well plates at 1×105 cells/well and pretreated with or without melatonin for 12 hand then stimulated for 12 h with 100 ng/mL LPS. The cells were then washed three times with DPBS, and fresh culture medium containing 5 mg/mL MTT (Sigma-Aldrich) was added to each well. After incubation at 37°C for 4 h, the culture medium was removed and 100 µL dimethyl sulfoxide (Nacalai Tesque) was added. The plates were oscillated for 15 min, and the absorbance at 570 nm was measured spectrophotometrically (Model 680 microplate reader S/N 22002; Bio-Rad Laboratories). Samples from three quails were analyzed in sextuplicate.
Model 680 microplate reader s n 22002
Manufactured by Bio-Rad
Sourced in United States
The Model 680 microplate reader S/N 22002 is a compact spectrophotometric device designed for microplate-based assays. It can measure the absorbance of samples in 96-well microplates across a wavelength range of 340 to 750 nm.
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4 protocols using model 680 microplate reader s n 22002
1
Granulosa Cell Viability Assay
2
Melatonin's Impact on bMEC Viability
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The viability of bMECs was quantified using an MTT assay. Briefly, cells were seeded in 96-well plates at 1 × 105 cells/well and incubated with or without melatonin (43 or 430 μM) for 12 h. Medium or 100 ng/mL LPS was then added and the cells were incubated for a further 12 h. The cells were washed three times with DPBS, and 5 mg/mL MTT (Sigma-Aldrich) in DPBS was added to each well. After 4 h incubation at 37°C, the supernatant was removed and 100 μL dimethyl sulfoxide (Nacalai Tesque) was added to stop the reaction. The plates were shaken for 15 min, and the absorbance at 570 nm was measured using a spectrophotometer (Model 680 microplate reader S/N 22002; Bio-Rad Laboratories). The measurements were performed in sextuplicate.
3
Nitrite Quantification in Cell Culture
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The level of the nitric oxide (NO) metabolite nitrite in samples of cell culture medium was measured with a nitrite colorimetric assay kit (Dojindo Molecular Technologies, Tokyo, Japan) based on the Griess reaction, according to the manufacturer's instruction. Samples were measured using a spectrophotometer (Model 680 microplate reader S/N 22002; Bio-Rad Laboratories, Hercules, CA, USA), and nitrite concentrations were calculated by comparison with a standard curve. Samples from six quails were analyzed in duplicate.
4
Measuring Nitrite Levels via Griess Assay
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Levels of the nitric oxide (NO) metabolite nitrite in samples of cell-free culture medium were measured with a nitrite colorimetric assay kit using the Griess reaction (Dojindo Molecular Technologies, Tokyo, Japan), according to the manufacturer's instructions. Samples were measured spectrophotometrically (Model 680 microplate reader S/N 22002; Bio-Rad Laboratories, Hercules, CA, USA) and nitrite concentrations (μM) were calculated with reference to a standard curve. Measurements were performed in duplicate.
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