The intracellular ROS level was determined using a MitoSOX™ Red mitochondrial superoxide indicator (Thermo Fisher Scientific, Invitrogen), a cationic derivative of dihydroethidium, to selectively examine the mitochondrial superoxide levels of live cells. In brief, cells were incubated with 5 μM MitoSOX™ Red reagent working solution in the dark at 37 °C for 30 min according to the manufacturer’s procedure. Then, the cells were washed 3 times with warm buffer. Then, the ROS signals were imaged with a fluorescence microscope (C1-T-SM, Nikon, Japan), and the fluorescence intensity was quantified using ImageJ software.
C1 t sm
Manufactured by Nikon
Sourced in Japan
The C1-T-SM is a compact confocal microscope system designed for live-cell imaging. It features a motorized stage and a sensitive detector for high-resolution imaging of fluorescent samples.
Automatically generated - may contain errors
Lab products found in correlation
F 2500, by Hitachi (3 mentions)
H2dcfda, by Thermo Fisher Scientific (2 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Jc 1 dye, by Thermo Fisher Scientific (1 mentions)
Dcfda cellular ros detection assay kit, by Abcam (1 mentions)
H2dcfda, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Bioss Antibodies (1 mentions)
6 protocols using c1 t sm
1
Mitochondrial Superoxide Quantification
2
Mitochondrial Membrane Potential Assay
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The level of the mitochondrial membrane potential (MMP) in cells was evaluated using JC-1 dye (Thermo Fisher Scientific, Invitrogen). In brief, the cells were collected and washed with PBS and incubated with JC-1 dye (5 mM) at 37 °C for 30 min. Then, the cells were washed and resuspended in PBS. Finally, the cells were imaged by a fluorescence microscope (C1-T-SM, Nikon, Japan), and the fluorescence intensity was analysed using Image J software.
3
ROS Detection in MCF-7 Cells
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Intracellular accumulation of ROS was estimated using the fluorescent dye H2-dichlorofluorescin diacetate (DCFDA; Life Technologies; Thermo Fisher Scientific, Inc.), which is converted to a membrane impermeable and highly fluorescent compound, dichlorofluorescin (DCF), in the presence of ROS. The MCF-7 cells were seeded in a 6-well plate at the density of 1×105 cells/well. Following treatment with the polysaccharide (100 μg/ml) or SP600125 (5 μM), MCF-7 cells (1×105 cells/well) were further incubated for 48 h. The cells were rinsed with serum-free medium and were incubated in 5 μM H2-DCFDA for 60 min at 37°C. The cells were then examined under a fluorescence microscope (C1-T-SM; Nikon Corporation, Tokyo, Japan), collected and subjected to a fluorescence spectrophotometry (F-2500; Hitachi, Ltd., Tokyo, Japan) to detect the DCF fluorescence inside cells (excitation, 488 nm; emission, 521 nm) as described (8 (link)).
4
Measuring Intracellular Reactive Oxygen Species
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The generation of intracellular ROS was assessed using 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA), a non-fluorescent probe, which upon oxidation by ROS and peroxides is converted to the highly fluorescent derivative dichlorofluorescin (DCF) [24 (link)]. We used a DCFDA cellular ROS detection assay kit (Abcam, Cambridge, MA, USA) as per the manufacturer’s instructions. In brief, an aliquot of the isolated cells (8 × 106 cells/mL) was made up to a final volume of 2 mL in normal PBS (pH 7.4). Then, a 1 mL aliquot of cells was taken to which 100 µL DCFDA (10 µmol/L) was added and incubated at 37 °C for 30 min and washed twice with PBS. Cells were solubilized in Triton-X100 1% (v/v) in distilled water. The cells were then examined under a fluorescence microscope (C1-T-SM; Nikon, Tokyo, Japan). The percentage of fluorescence-positive cells was measured at an excitation wavelength of 488 nm and an emission wavelength of 535 nm by a fluorescence spectrophotometer (F-2500; Hitachi, Tokyo, Japan).
5
ROS Accumulation Measurement in A375 Cells
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Intracellular accumulation of ROS was estimated using the fluorescent dye H2-DCFDA (Thermo Fisher Scientific, Inc.) as described previously (20 (link)). The A375 cells were seeded at a density of 1×105 cells/well, in a six-well plate, with or without PDTC (100 µmol) pretreatment. Following transfection with CDK5RAP1 siRNA or control siRNA for 48 h, A375 cells were washed with serum-free DMEM medium (Sigma-Aldrich; Merck KGaA) and incubated in H2-DCFDA (5 µM) for 60 min at 37°C. The cells were then examined under a fluorescence microscope (C1-T-SM; Nikon Corporation, Tokyo, Japan). Finally, cells were collected and subjected to fluorescence Spectrophotometer (F-2500; Hitachi, Ltd., Tokyo, Japan) to detect the fluorescence of DCF inside cells (excitation, 488 nm; emission, 521 nm).
6
CDK5RAP1 Silencing in A375 Cells
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A375 cells were plated in six-well plates at a density of 1×105 cells/well and pretreated with or without PDTC (100 µmol). Cells were washed with PBS after transfection with CDK5RAP1 siRNA or control siRNA for 48 h. Cells were then fixed in 4% paraformaldehyde (BIOSS, Beijing, China) for 30 min at 22°C followed by staining with Hoechst 33342 (20 mg/ml) for 15 min at room temperature in the dark. Cells were then imaged by using fluorescence microscopy (C1-T-SM; Nikon Corporation, Tokyo, Japan) at a magnification, ×100.
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