Tumor cells were seeded at a density of 5 × 104 cells/ml in ultra-low attachment 6 well plates with EMEM medium 10% FBS. On top of the well, co-culture was performed in a transwell insert with 0.4 μm membrane seeded with HMC3 cells (105 cells per well) or with 50:50 HMC3:tumor cells ratio. Two days after co-culture, Reactive Oxygen Species (ROS) level in cells seeded in lower chamber was evaluated. Cells were incubated with 10 μM of CellRox Orange dye (Invitrogen) at 37°C for 30 min. To verify the specificity of the CellRox reagent, ROS was activated by treatment with 50 μM Luperox® TBH70X just before cell staining. In some experiments, co-cultures were incubated with 5mM N-Aceytyl-L-cysteine. 7-AAD was added to exclude dead cells during recording. Fluorescent cells were detected by FACS ARIAII.
Cellrox orange dye
Manufactured by Thermo Fisher Scientific
CellRox Orange dye is a fluorescent probe used to measure oxidative stress in live cells. It detects the presence of reactive oxygen species (ROS) and can be used with various cell types and experimental conditions.
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Lab products found in correlation
2 protocols using cellrox orange dye
1
ROS Evaluation in Tumor-Microglia Co-culture
2
Seminal Vesicle ROS Quantification
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After paternal diet intervention for 2 days, seminal vesicles were dissected (from Dj-GFP or W1118) in insect medium (Sigma; # T3160) (for CellROX) or 1 × PBS (H2DCFDA), and they were subsequently incubated in CellROX orange dye (Invitrogen; # C10443) for 30 min at 37°C (5 μM final concentration in insect medium) or H2DCFDA (Invitrogen #D399) at room temperature for 5 min (40 μM final concentration). After this, the tissue was rinsed extensively with 1 × PBS; mounted onto glass slides with halocarbon oil (HC700; Sigma) that served as the mounting medium; and covered with glass coverslips.
Coverslips were sealed with clear nail polish, and slides were imaged using an inverted confocal microscope (LSM800; Zeiss) using absorption/emission maxima of ∼545/565 nm (for CellROX) or ∼488/517 nm (for H2DCFDA and GFP). The timing of start of dietary regimen, and that of imaging for ROS was kept constant across all experiments. All images were quantified in Fiji (Schindelin et al, 2012 (link)). The steps followed to process the images in Fiji are depicted next. The mean gray values are plotted in graphs.
Coverslips were sealed with clear nail polish, and slides were imaged using an inverted confocal microscope (LSM800; Zeiss) using absorption/emission maxima of ∼545/565 nm (for CellROX) or ∼488/517 nm (for H2DCFDA and GFP). The timing of start of dietary regimen, and that of imaging for ROS was kept constant across all experiments. All images were quantified in Fiji (Schindelin et al, 2012 (link)). The steps followed to process the images in Fiji are depicted next. The mean gray values are plotted in graphs.
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