Cells were seeded in 6-well plates (H1581: 1 × 105 cells/mL; KATO III: 1 × 105 cells/mL; RT-112: 6 × 104 cells/mL) and allowed to adapt overnight. The cells were incubated with pemigatinib (100 nM) for 48 h. Untreated cells (NT) were used as experimental controls. Cells treated with H2O2 for 1 h in 37 °C 5% CO2 incubator were used as positive control. After treatment, cells were washed with PBS, and the adherent cells (KATO III and RT-112) were immediately incubated with 10 mM DCFDA/H2DCFDA (Abcam, ab113851) for 15 min in an incubator. Then, the cells were washed and harvested with trypsin. H1581 cells were washed and then incubated with 10 mM DCFDA/H2DCFDA (Abcam) in a 5 mL FACStube (Falcon, 352054) and then washed. The analysis was performed using FACSCanto II and FlowJo Software (BD, Biosciences). For the immunofluorescence assay, the cells were seeded on a coverslip and after probe incubation were analyzed, employing Apotome Microscope (40X magnification). The results were derived from three independent experiments.
Dcfda h2dcfda
Manufactured by Abcam
Sourced in United Kingdom
DCFDA/H2DCFDA is a fluorescent dye used to detect and measure oxidative stress in cells. It is a cell-permeant indicator for reactive oxygen species (ROS) that fluoresces when oxidized.
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Lab products found in correlation
Fluorescence microscope, by Olympus (2 mentions)
Facscanto 2, by BD (1 mentions)
Facs tube, by BD (1 mentions)
Victor x3 light plate reader, by PerkinElmer (1 mentions)
Nhek ad, by Lonza (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Mito tracker green, by Beyotime (1 mentions)
Cellrox green reagent, by Thermo Fisher Scientific (1 mentions)
Novocyte flow cytometer, by Agilent Technologies (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti erk1 2 3a7, by Cell Signaling Technology (1 mentions)
Anti ps473 akt, by Cell Signaling Technology (1 mentions)
Cellular ros assay kit deep red, by Abcam (1 mentions)
Ix70 inverted microscope, by Olympus (1 mentions)
Cellular ros assay kit, by Abcam (1 mentions)
6 protocols using dcfda h2dcfda
1
Pemigatinib-Induced Oxidative Stress Assay
2
UV-Induced ROS Measurement in NHEK
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Human adult primary keratinocytes (NHEK-Ad, Lonza) were plated on black 96-well cell culture plates (SPL). When cells reached 70–80% confluence, 20 μL DCFDA/H2DCFDA (Abcam; Cambridge, UK) was pre-loaded to each well followed by UV exposure. The fluorescence intensity of each well was measured 30 min after irradiation with excitation 485 nm and emission 530 nm, using a Victor X3 light plate reader (Perkin-Elmer) or observed immediately under a fluorescence microscope (Olympus; Tokyo, Japan).
3
Intracellular ROS Regulation in LX-2 Cells
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Intracellular reactive oxygen species (ROS) in LX-2 cells treated with succinic acid (1,600 μmol/L) with or without gemigliptin, and 5 mM NAC for 1 hour were analyzed using DCFDA/H2DCFDA (Abcam, Cambridge, UK, ab113851). After treatment for 1 hour with reagents, LX-2 cells were stained with DCFDA (20 μM) for 45 minutes at 37°C in the dark. Images were obtained using a fluorescence microscope (Olympus, Tokyo, Japan).
Mitochondrial ROS production was examined using the superoxide indicator MitoSOX Red (M36008, Invitrogen). LX-2 cells were plated and treated with succinic acid (1,600 μmol/L) with or without gemigliptin for 1 hour. Next, the cells were washed with phosphate-buffered saline and then incubated with 5 μM MitoSOX reagent and MitoTracker Green (Beyotime, Shanghai, China) for 30 minutes at 37°C in the dark. Images were obtained using a confocal scanning microscope (Olympus).
Mitochondrial ROS production was examined using the superoxide indicator MitoSOX Red (M36008, Invitrogen). LX-2 cells were plated and treated with succinic acid (1,600 μmol/L) with or without gemigliptin for 1 hour. Next, the cells were washed with phosphate-buffered saline and then incubated with 5 μM MitoSOX reagent and MitoTracker Green (Beyotime, Shanghai, China) for 30 minutes at 37°C in the dark. Images were obtained using a confocal scanning microscope (Olympus).
4
Cellular ROS Level Quantification
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To determine the cellular ROS level, cells grown to confluence were stained with CellROX™ Green Reagent (Invitrogen) at a final concentration of 2.5 μmol/L and incubated for 30 minutes at 37°C for MCF12A, and 10 μmol/L with 1-hour incubation at 37°C for MDA-MB-231, which were then washed with PBS, trypsinized, and subjected to FACS analysis by NovoCyte Flow Cytometer (ACEA Biosciences, San Diego, CA, USA). On the other hand, cells in suspension were stained with DCFDA/H2DCFDA (Abcam, Cambridge, UK) at a final concentration of 1 μmol/L and incubated for 15 minutes at 37°C for MCF12A, and 4 μmol/L with 15-minute incubation at 37°C for MDA-MB-231, which were then washed and subjected to FACS analysis.
5
Immunoblotting and ROS Assay Protocol
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SDS-PAGE and immunoblotting were performed as described earlier [25 (link)]. The following antibodies were used: anti-pY694 STAT5 (ab32364, Abcam, Cambridge, UK); anti-pY589/591 FLT3 (#3464), anti-pS473 AKT (#4060), anti-pERK1/2 137F5 (#4695), anti-AKT (#9272), and anti-ERK1/2 3A7 (#9107) from Cell Signaling (Frankfurt, Germany); anti-FLT3 (sc-480) and anti-STAT5a/b rabbit polyclonal (sc-835) from Santa Cruz (Heidelberg, Germany).
Reactive oxygen species were determined using the dye DCFDA/H2DCFDA (ab113851, Abcam, Cambridge, UK) or the Cellular ROS Assay Kit (Deep Red) (ab186029, Abcam, Cambridge, UK) and subsequent FACS analysis according to the instructions of the manufacturer.
Reactive oxygen species were determined using the dye DCFDA/H2DCFDA (ab113851, Abcam, Cambridge, UK) or the Cellular ROS Assay Kit (Deep Red) (ab186029, Abcam, Cambridge, UK) and subsequent FACS analysis according to the instructions of the manufacturer.
6
Cellular Oxidative Stress Assay
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Cells (1 × 104) were seeded in black 96-well plates and cultivated as previously described. We used the 2’,7’-dichlorofluorescein diacetate/2’,7’-dichlorodihydrofluorescein diacetate (DCFDA/H2DCFDA) − Cellular ROS Assay Kit (Abcam, UK) according to the manufacturer’s instructions. Measurements were performed at 3 and 24 hpi by fluorescence quantification of each cell using ImageJ v.1.53m software (NIH) from at least four images per group (40× magnification) using an Olympus IX70 inverted microscope. Data are presented as corrected total cell fluorescence (CTCF) calculated as integrated density – (area of selected cell × mean fluorescence of background readings). Analyses were performed using the ratio of cell CTCF and M0 mean CTCF.
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