For detection of cellular ROS, cells were seeded 25 × 103 cells per well on a 96-well plate and received either control, 1 or 10 mM NaAc treatment for 15 min or 24 h. Then cells were stained with 2′,7′-dichlorofluorescin diacetate (DCFDA) using DCFDA Cellular ROS Detection Kit according to the manufacturer's instructions (Abcam, USA) and read on a fluorescent plate reader (ex/em: 495/529 nm) (FLUOstar Optima).
Dcfda cellular ros detection kit
Manufactured by Abcam
Sourced in United Kingdom
The DCFDA Cellular ROS Detection Kit is a fluorescent probe-based assay used to detect and quantify the levels of reactive oxygen species (ROS) in cells. The kit utilizes 2',7'-dichlorofluorescin diacetate (DCFDA) as the fluorescent indicator, which is oxidized by ROS to produce the highly fluorescent 2',7'-dichlorofluorescein (DCF). The intensity of the fluorescent signal is proportional to the levels of ROS present in the sample.
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12 protocols using dcfda cellular ros detection kit
1
Cellular ROS Detection Assay
2
Intracellular ROS Measurement in BV-2 Cells
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Intracellular reactive oxygen species (ROS) levels were measured using the DCFDA cellular ROS detection kit (Abcam, Cambridge, UK). After internalization and subsequent hydrolysis, the redox indicator probe carboxy-H2DCFDA is converted to carboxy-H2DCF, which, in the presence of oxidant species, is converted to fluorescent carboxy-DCF [57 (link)]. BV-2 cells were seeded in black clear-bottom 96-well plates at a density of 5 × 104 cells/well [43 (link)]. Cells were allowed to adhere overnight and then incubated with 20 μM DCFDA for 40 min at 37 °C in the dark. The solution was removed, and the cells were incubated in serum-free medium, containing LPA in the absence or presence of the antagonists for 3 and 6 h. Fluorescence intensity was measured with excitation and emission wavelengths of 485 and 535 nm, respectively.
3
Intracellular Oxidative Stress Assay
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Intracellular oxidative stress was evaluated based on detection of fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) (DCFDA cellular ROS detection kit, Abcam, Cambridge, UK) using a microplate reader (BioTek HT, BioTek Instrument GmbH, Bad Friedrichshall, Germany). The 16HBE cells were seeded at 104 cells/well density into a 96-well black-walled plate with transparent bottom (Greiner Bio-One GmbH, Frickenhausen, Germany) and incubated for 3 days at the humidified incubator with 7.5% CO2. Next, the cells were washed once with diluted assay buffer and stained with 25 µM of DCFDA diluted in an assay buffer for 45 min. The cells were then stimulated with different concentrations of CSE (5–20%), medium control or a ROS-positive control compound tBHP, diluted in the complete serum-free medium. The fluorescent signal was initially measured at 0 h time before stimulating the cells by exciting at 488 nm and emitting at 528 nm (FITC filter set) and a PMT gain set at 45. Afterwards, the plate kept in the incubator and read at 1 h, 3 h, 6 h, 19 h, 21 h, 23 h, 25 h, and 28 h post treatment. The fluorescent signal was calculated by subtracting the unstained background and normalizing the signals from each condition to 0 h.
4
Cellular ROS Quantification via DCFDA
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Cellular ROS was assessed using the 2′,7′-dichlorofluorescein diacetate (DCFDA) assay with a microplate reader [18 (link)]. In this study, the DCFDA assay was performed according to the manufacturer’s instructions (DCFDA Cellular ROS Detection Kit, Abcam, Cambridge, UK). In brief, the cells were incubated with 10 μL of DCFDA for 30 min in the dark and washed out with PBS. Accumulation of the oxidized fluorescent derivate (DCF) was measured using flow cytometry at an excitation/emission wavelength of 485/530 nm.
5
Oxidative Stress Analysis in SH-SY5Y Cells
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Following treatment in one of the manners described above, SH‐SY5Y cells were homogenized and the supernatant was prepared by centrifugation at 4°C. The level of MDA and activity of SOD were determined using commercial kits (Nanjing Jiancheng Bioengineering Int.), while ROS was measured using a DCFDA‐cellular ROS detection kit (Abcam).
6
DCFDA-based Cellular ROS Assay
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For detecting ROS levels, a DCFDA-cellular ROS detection kit (Abcam) was used. Briefly, cells were incubated with 20 μM DCFDA for 1 h at 37 °C, and administration of 500 nM TBHP (2 h at 37 °C) was used as a positive control. All cells were collected without further washes as suggested by the manufacture’s protocol for flow cytometry analysis.
7
Measuring ROS Generation in HMEC-1
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Reactive oxygen species (ROS) generation by HMEC-1, assessed in cell supernatants after treatment with AHE, was performed using 2′,7′-dichlorofluorescin diacetate (DCFDA)-ROS detection kit (DCFDA Cellular ROS Detection Kit, Abcam, UK) according to manufacturer instructions. Results are expressed as means of fluorescence units ± SD.
8
Quantifying Intracellular Reactive Oxygen Species
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The production of free radicals was assessed by using DCFDA (2′,7′-dichlorofluorescein diacetate), which is one of the most widely used techniques for direct measuring of cell redox state [37 (link)]. DCFDA is a fluorogenic dye for highly selective detection of hydroxyl, peroxyl, and any other intracellular reactive oxygen species (ROS) activity. DCFDA is diffused into cells and is deacetylated by cellular esterases into a non-fluorescent compound that is subsequently oxidized by ROS into DCF (2′,7′-dichlorofluorescein). DCF is a highly fluorescent compound and hence is detectable by fluorescence spectroscopy.
The DCFDA assay was performed according to the manufacturer’s instruction (DCFDA Cellular ROS Detection Kit, Abcam, Cambridge, UK). In brief, 10 μL of DCFDA was added to cells and incubated for 30 min. After being washed out with PBS, the intensity of fluorescence was examined by flow cytometry. Accumulation of the oxidized fluorescent derivate (DCF) in the cells was measured at emission and excitation wavelengths of 530 and 485 nm, respectively.
The DCFDA assay was performed according to the manufacturer’s instruction (DCFDA Cellular ROS Detection Kit, Abcam, Cambridge, UK). In brief, 10 μL of DCFDA was added to cells and incubated for 30 min. After being washed out with PBS, the intensity of fluorescence was examined by flow cytometry. Accumulation of the oxidized fluorescent derivate (DCF) in the cells was measured at emission and excitation wavelengths of 530 and 485 nm, respectively.
9
Intracellular ROS Detection via DCFDA
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Intracellular reactive oxygen species (ROS) levels were measured using the 2′,7′-dichlorofluorescin diacetate (DCFDA) cellular ROS detection kit (Abcam, Cambridge, UK). After internalization and subsequent hydrolysis, the redox indicator probe carboxy-H2DCFDA is converted to carboxy-H2DCF, which in the presence of oxidant species is oxidized to fluorescent carboxy-DCF [46 (link)]. BV-2 and PMM were seeded in black clear bottom 96-well plates at a density of 2.5 × 104 cells per well. Cells were allowed to adhere overnight and then incubated with 20 μM DCFDA for 40 min at 37 °C in the dark. The solution was removed and the cells were treated with vehicle control (DMSO), LPA, or LPA plus TCLPA5 for 0.5, 1, 3, and 6 h. Fluorescence intensity was measured with excitation and emission wavelengths of 485 and 535 nm, respectively.
10
Measurement of Reactive Oxygen Species in MIN6 Cells
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ROS detection was performed using a DCFDA cellular ROS detection kit (Abcam). Mixing individual hIAPP species with DCFDA abiotically did not show any effect on the dye fluorescence in aqueous solution (Supplementary Figure S1 ). Single cell suspensions of MIN6 cells were stained with 200 nM of DCFDA for 30 min and subsequently treated with fresh hIAPP, stabilised oligomeric hIAPP and mature amyloid for 2 h and 4 h to avoid death of control cells in suspension. ROS levels were then measured indirectly by the oxidation of nonfluorescent DCFDA to fluorescent DCF by a flow cytometer, exciting the dye at 488 nm and detection at 535 nm. The percentage of ROS positive cells was then quantified using FlowJo Software, analysing the ROShi (DCF+) population.
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