For intracellular ROS measurements, cells cultured under attachment or detachment conditions were incubated with 25 mM of carboxy-H2DCFDA (Sigma, St. Louis, MO, USA) for 30 min at 37 °C, harvested, and analyzed by FCM on a flow cytometer (Becton Dickinson Bioscience, San Jose, CA, USA).
Carboxy h2dcfda
Manufactured by Merck Group
Sourced in United States
Carboxy-H2DCFDA is a fluorogenic dye used to measure intracellular oxidative stress and reactive oxygen species in live cells. It undergoes an oxidation-sensitive change in fluorescence upon reaction with reactive oxygen species.
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Lab products found in correlation
Fluorescence microscope, by Nikon (2 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Bullet blender, by Next Advance (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
H2dcfda, by Merck Group (1 mentions)
Enspire multiplate reader, by PerkinElmer (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Dcf 2 7 dichlorofluorescein assay, by Thermo Fisher Scientific (1 mentions)
Incyte software, by Merck Group (1 mentions)
Enspire multimode plate reader, by PerkinElmer (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Spectramax microplate reader, by Molecular Devices (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Bricyte e6 flow cytometer, by Mindray (1 mentions)
Varioskan lux, by Thermo Fisher Scientific (1 mentions)
Hydroxyphenyl fluorescein solution, by Merck Group (1 mentions)
Axiovert 200m inverted microscope, by Zeiss (1 mentions)
Facscanto, by BD (1 mentions)
Anti cd11b pe cy7, by BD (1 mentions)
Anti cd66b fitc, by BD (1 mentions)
18 protocols using carboxy h2dcfda
1
Intracellular ROS Measurement Protocol
2
Quantification of Cellular Reactive Oxygen Species
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Measurement of ROS was performed as previously reported [64 ]. Briefly, samples were the same as the above iNOS detection. Samples were lysed using a bullet-blender (Bullet Blender, Next Advance, USA) and then centrifuged at 12,000 g for 10 min. After centrifugation, supernatants were collected and protein concentrations were determined by Coomassie-blue method. For ROS quantification by fluorescence, samples were incubated with 10 mM carboxy-H2DCFDA (Sigma, USA) at 37°C for 30 min in the dark and analyzed by a microplate reader (BioTek, synergy 2, USA) at an excitation and emission wavelength of 495 and 525 nm, respectively.
3
Measuring Oxidative Stress in B16-BL6 Cells
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Oxidative stress was determined by measuring the production of ROS using 5-(and-6)-carboxy-2′, 7′-dichlorodihydro-fluorescein diacetate (carboxy-H2DCFDA) (Sigma, St. Louis, MO) The nonpolar, nonionic H2-DCFDA is cell permeable and is hydrolyzed to nonfluorescent H2-DCF by intracellular esterases. H2-DCF is rapidly oxidized to highly fluorescent DCF when the peroxide presenting. Briefly, 1.2 ×105 B16-BL6 cells were seeded in a 12-well plate and allowed to grow overnight. Cells were transfected with GL2 siRNA or IDO2 siRNA for 48 h and then harvested, pelleted by centrifugation, re-suspended with 5μl MH2DCFDA in DMSO for 30 min. Cells were detected using a BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ) and analyzed by FlowJo software (Tree Star, Inc., Ashland, OR, USA).
4
Intracellular ROS Measurement in Cells
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The level of the ROS in cells was determined using the DCF (2,7-dichlorofluorescein) assay (Life Technologies, Poland). For experiments, the stock solution of carboxy-H2DCFDA (50 µg/ml in sterile DMSO; Sigma, Poland) was established at the RT in the dark and then diluted in a cell culture medium without FBS. The cells were seeded on black 96-well plates (3 × 104 cells/cm2). After washing with PBS, the reagent was added to the cell culture to a final concentration of 5 µM, and cells were incubated at 37°C in darkness for 30 min. After the incubation, the mean fluorescence of DCF in wells was measured every 30 min for 90 min total using a plate reader (Multiplate Reader EnSpire, Perkin Elmer) with excitation wavelength of 495 nm and emission wavelength of 530 nm. Intracellular ROS generation assay was evaluated directly after balloon flight (0 h) and 24 h after landing (24 h).
5
ROS Detection via Carboxy-H2DCFDA Staining
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Cells (2 × 105) were seeded in 6-well plates and then treated by using the coincubation or preincubation scheme. The cells were later washed with phosphate-buffered saline solution (PBS) and harvested with PBS-EDTA, followed by centrifuging at 2,000 rpm for 8 minutes and resuspension in PBS. For ROS detection, they were treated with 10 μM of the fluorescent marker 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA, Sigma-Aldrich) [21 (link)] for 30 minutes in the dark. Flow cytometry was performed over 10,000 acquired events with InCyte software (Merck Millipore, Darmstadt, DE), with hydrogen peroxide (1 mM per 24 h) used as the positive control. The experiment was carried out in triplicate.
6
Measuring Intracellular ROS Levels
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The level of intracellular ROS was measured by 5-(and-6)-carboxy-2′,7′-dichiorodihydroflurescein diacetate (carboxy-H2DCF-DA, Sigma) as reported in our previous publication [20 (link)]. Briefly, cells were washed once with ice-cold phosphate-buffered saline and incubated with 10 μM carboxy-H2DCF-DA at 37°C for 10 min. Cells were then washed once with ice-cold phosphate-buffered saline and scanned with a plate reader at 485 nm excitation and 520 nm emission. Images were acquired by a fluorescence microscope (Nikon). Unless otherwise indicated, the fluorescence intensity in SH-SY5Y cells without treatment is expressed as a percentage of the control.
7
Evaluating Antioxidant Activity of rhSLPI
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ARVMs were cultured with modified M199 medium in 24-well black plates and maintained at 37°C in an atmosphere containing 5% CO2 and 95% O2. The culture medium was removed and the cells were washed with phosphate-buffered saline (PBS). Subsequently, cells were incubated with M199 medium containing 25 µM carboxy-H2DCFDA (Sigma-Aldrich; Merck KGaA) in a dark room for 30 min at 37°C. The medium containing carboxy-H2DCFDA was discarded and ARVMs were washed once with PBS. For rhSLPI treatment, 500 µl M199 medium containing various concentrations of rhSLPI (0, 200, 400, 600, 800 and 1,000 ng/ml) was added and incubated for 1 h at 37°C. Subsequently, cells were incubated with 250 µM H2O2 for 30 min at 37°C. The control cells were incubated with M199 medium without treatment. ROS activity was determined by measuring fluorescence intensity using an EnSpire Multimode Plate Reader (PerkinElmer, Inc., Waltham, MA, USA) at an excitation wavelength of 498 nm and emission wavelength of 522 nm.
8
ROS Measurement in H460 Cells
9
Measuring Oxidative Stress in E. coli
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To detect ROS formation by means of flow cytometry analysis, cultures of E. coli CC118 transformed with vector pSEVA237M were incubated overnight as indicated. The following day, samples were diluted in 25 ml of filtered LB medium to an initial OD600 ~ 0.05. At the mid‐exponential phase of growth (OD600 ≈ 0.4), the culture was divided into 7 ml aliquots that were treated either with the stressors H2O2 or diamide (as positive controls of oxidative stress) or with 1 mM of each of the inducers under inspection. One more aliquot was kept untreated as negative control. After an additional 2 h incubation, 1 ml of the cultures was spun down at 13 000 g for 2 min at room temperature, washed once with 1 ml of filtered phosphate buffered saline by centrifugation and resuspension and diluted in filtered phosphate buffered saline such that the OD600 was under 0.4 in a final volume of 1 ml. Then, a freshly prepared solution of 5‐(6)‐carboxy‐2′,7′‐dichlorodihydrofluorescein diacetate (carboxy‐H2DCFDA; Sigma‐Aldrich, St. Louis, MO, USA) in DMSO was added to the samples at 20 μM. Cell suspensions were incubated for 30 min at 37°C protected from light. Finally, the fluorescence of each sample was measured in the green channel using the same excitation/emission parameters of msfGFP.
10
Quantifying Intracellular ROS in Kidney Samples
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Two kinds of fluorescent probes were used to detect intracellular ROS in kidney tissues and cells, namely 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA; Sigma-Aldrich), and dihydroethidium (DHE; Sigma-Aldrich). For DHE staining, cryosections were incubated with 10 μM DHE at 37 °C for 30 min and then observed by confocal microscopy to determine the percentage of the DHE-stained area. For carboxy-H2DCFDA staining, kidney tissues were homogenized with PBS and centrifuged. Then, the supernatant was collected and incubated with 10 μM H2DCFDA at 37 °C for 30 min. For ROS detection in cultured cells, the cells were seeded in a 96-well plate and incubated with H2DCFDA. Fluorescence was detected by a SpectraMax Microplate Reader (Molecular Devices, San Jose, CA) at 488 nm excitation and 525 nm emission.
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