Intracellular accumulation of ROS was measured using a dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay (Nanjing KeyGen Biotech). Cells were treated with DMC (40 µmol/L), UVB (50 mJ/cm 2 ), DMC-PDT (40 µmol/L and 50 mJ/cm 2 , respectively; DMC was precultured for 12 h before UVB irradiation), or DMC-PDT-N-acetyl-l-cysteine (NAC; 40 µmol/L, 50 mJ/cm 2 , and 5 mmol/L, respectively). The UVB irradiation occurred following DMC preincubation. NAC was added 2 h prior to irradiation. Following treatment, cells were harvested, washed twice with PBS, and incubated with 10 µmol/L DCFH-DA for 20 min. Cells were washed three times with PBS, resuspended in 0.5 mL PBS, and analyzed by FCM for ROS changes.
Dcfh da
Manufactured by Keygen Biotech
Sourced in China
DCFH-DA is a fluorescent probe used to detect the presence of reactive oxygen species (ROS) in biological systems. It is a non-fluorescent compound that can cross cell membranes and is converted to a fluorescent product upon oxidation by ROS.
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Lab products found in correlation
Cytoflex, by Beckman Coulter (2 mentions)
Cantoii, by BD (1 mentions)
A fluorescent microscope, by Zeiss (1 mentions)
Oxiselect nitrotyrosine kit, by Cell Biolabs (1 mentions)
Fortessa system, by BD (1 mentions)
Flow cytometry, by BD (1 mentions)
Facscalibur flow cytometry, by BD (1 mentions)
N acetyl cysteine (nac), by Beyotime (1 mentions)
Dihydroethidium dhe, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscopy, by Olympus (1 mentions)
Facsaria 2, by BD (1 mentions)
C11 bodipy581 591, by Thermo Fisher Scientific (1 mentions)
Staurosporine, by Bio-Techne (1 mentions)
Nec 1, by Merck Group (1 mentions)
Mouse anti β actin antibody, by Abcam (1 mentions)
Matrine, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
N acetyl l cysteine, by Merck Group (1 mentions)
Z vad fmk, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Keygen Biotech (1 mentions)
38 protocols using dcfh da
1
ROS Measurement in Photodynamic Therapy
2
Compound 968 Induces Oxidative Stress in Endometrial Cancer
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The ability of the compound 968 to affect ROS generation and induce cellular oxidative stress in human endometrial cancer cells was assessed by measuring the intracellular ROS levels using the ROS-sensitive uorescence indicator DCFH-DA (Jiangsu KeyGEN BioTECH Corp. Ltd, Jiangsu, China) and ow cytometry, as described previously (Eruslanov E and Kusmartev S, 2010). In brief, Ishikawa and HEC-1B cells were seeded in 6-well plates at 2.5 × 10 5 cells/well. After cells were allowed to adhere for 5 h, the cells were treated with 5, 10, and 25 μM of compound 968 for 12 h. Subsequently, cells were incubated in the presence of DCFH-DA (20 μM) for 20 min, and then harvested, washed with PBS, and then analyzed on the FACS Calibur instrument. The ow cytometry results were analyzed using the FlowJo Software, version 7.6. At least two independent repetitions were performed for each condition.
3
Oxidative Stress Assessment via DCFH-DA
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Cells were seeded into a 24‐well plate at a density of 3 × 103/well and exposed to different processing conditions. After washing three times with PBS, the centrifuged cells were collected, and the cell pellet was suspended in a concentration of 10 μm DCFH‐DA (KeyGEN Biotech, Nanjing, China). Following 30 min of incubation at room temperature in the dark, flow cytometry was used to analyze the average fluorescence intensity.
4
Measuring Cellular ROS Levels
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The differently-treated H9c2 cells were collected and washed with pre-cooled PBS. After loading with the probe DCFH-DA (10 M, KeyGEN BioTECH, Nanjing, China), the remaining probes that did not enter the cells were washed, and the total ROS level was detected using the flow cytometer (Becton Dickinson, Heidelberg, Germany).
5
Intracellular ROS Measurement by Flow Cytometry
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Intracellular ROS levels were detected by flow cytometry using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). HepG2 cells were washed three times with PBS solution, and cultured in serum-free DMEM containing DCFH-DA (100 µM) for 30 min in the dark at 37˚C. Cells were then washed in serum-free DMEM and treated with pancreatic enzymes. DCF fluorescence was detected at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.
6
ROS Modulation by BBR Analogues
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HCT-8 cells were cultured with RPMI 1640 medium containing 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL), and maintained at 37°C in humidified CO2 (5%). The cells in logarithmic growth phase were digested with 0.25% trypsin, added to RPMI 1640 medium supplemented with 10% FBS, plated into 6-well plates (106 cells per well), and incubated for 8 hrs. The cells were treated with BBR (50 μM) or 13-Cys-BBR (50 μM) for 6 hrs, into each well 200 μL of DCFH-DA (KeyGENBioTECH, KGAF019, diluted 800 times with RPMI 1640 medium) were added, then digested with 0.25% trypsin and collected into a 1.5mL-tube. After centrifugation (1000 g per min, 2.5 mins), the supernatant was removed, the residue was washed with PBS for 2 times, and then tested on flow cytometry and observed by fluorescence microscope (Beckman CytoFLEX).23 (link)
7
Intracellular ROS Measurement via DCFH-DA
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Intracellular ROS generation was detected using 2', 7'-dichlorofluorescin diacetate (DCFH-DA) (KeyGEN Biotech, China). For measurement of intracellular ROS levels, cells were incubated for 15 min with 2.5 µmol/ml DCFH2-DA at 37ºC for 30 min in the dark. After two washes with PBS, the cells were analyzed using a fluorescence microscope (Olympus BX 51, Tokyo, Japan). Cells with green fluorescence were considered ROS-positive cells. The mean fluorescence density of ROS generation was captured using a fluorescence microscope (Olympus BX51, Olympus, Tokyo, Japan) and processed using Image J software (NIH). At least 50 cells were randomly selected from a single captured field, and the average nuclear fluorescence intensity was calculated. Data points presented in the text are the averages calculated from five different fields..
8
Evaluating Intracellular ROS Levels
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Cells were treated with BUP for 48 h at distinct doses (40, 60, and 80 μM). The 0.1% DMSO-treated cells were used as a control. Then, the DCFH-DA (Keygen, Nanjing, China) was used to analyse intracellular ROS generation [23 (link)]. Cells were harvested by centrifuge (2000× g, 3 min) and washed two times with PBS, then hatched with 10 μM DCFH-DA for 30 min at 37 °C. The DCFH-DA was hydrolysed to DCFH and then oxidized to DCF. Its green fluorescence was detected using flow cytometry (BD CantoⅡ, Franklin Lakes, NJ, USA). The statistics of increased ROS level were calculated using the DCF fluorescence-positive cell ratio by FlowJo (V10, 2015) software (BD, Ashland, OR, USA).
9
ROS Detection in Cardiac Fibroblasts
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The sensitive ROS fluorescent probe-DCFH-DA (KGT010-1; KeyGEN BioTECH, Nanjing, China) was used to detect the ROS production following the manufacturer's instructions. In brief, cardiac fibroblasts incubated with 5 µM DCFH-DA at 37°C for 30 min. The fluorescence was then detected on flow cytometer (Becton Dickinson Biosciences, Franklin Lakes, NJ).
10
ROS Detection in AD-MSCs using DCFH-DA
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The sensitive ROS fluorescent probe-DCFH-DA (KGT010-1; KeyGEN BioTECH, Nanjing, China) was used to detect the ROS accumulation following the manufacturer’s instructions. In brief, AD-MSCs were incubated with 5 μM DCFH-DA at 37°C for 30 min. The fluorescence was then detected on flow cytometer (Becton Dickinson Biosciences, Franklin Lakes, NJ).
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