Cell viability of NOX4-overexpressing and negative-control RKO cells was evaluated using the cell counting kit-8 (CCK8, Dojindo Molecular Technologies, Japan). Briefly, the RKO cells were seeded into 96-well plates at a density of 5 × 103 cells/well. CCK8 solution was added to each well at 24, 48, and 72 h, and cells were incubated for 30 min at 37 °C. Absorbance at 450 nm was measured using a microplate reader (spectraMax iD3, PerkinElmer, United States). The cell cycle was examined using propidium iodide (PI) (BD Biosciences) staining. Cells were washed with cold phosphate buffered saline (PBS), fixed in 75% ethanol at -20 °C overnight, incubated with PI for 15 min, and then analyzed using flow cytometry.
Spectramax id3
Manufactured by PerkinElmer
Sourced in United States
The SpectraMax iD3 is a multi-mode microplate reader from PerkinElmer. It is designed to perform various absorbance, fluorescence, and luminescence assays in a 96- or 384-well microplate format. The SpectraMax iD3 can be used for a wide range of applications, including cell-based assays, protein and nucleic acid quantification, and enzyme activity measurements.
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Lab products found in correlation
2 protocols using spectramax id3
1
Cell Viability and Cell Cycle Analysis
2
Cytotoxicity Assay with Luc-Raji Cells
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Cytotoxicity were tested using luciferase‐labelled target cells (Luc‐Raji) as described by Melaiu et al.35 Luc‐Raji cells were suspended in RPMI‐1640 medium supplemented with 10% FBS in 96‐well, flat‐bottomed white view plates (Corning). Expanded NK cells suspended in RPMI 1640 with 10% FBS and 400 IU/ml IL‐2 were added at varying effector to target cell ratios as indicated in results section. The plates were incubated for 4 h at 37°C with 5% CO2. At the end of the culture, cell mixture was incubated with equal volume of D‐luciferin bioluminescent substrate (Perkin‐Elmer, Waltham, USA) for 10 min, then measured using a microplate reader (SpectraMax iD3). Viability was calculated by comparing relative luminescent signal from control wells on each plate. All experiments were done in triplicates.
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