To evaluate F. oxysporum oxidative stress generated by NA (10 mM) and formaldehyde (10 mM), reactive oxygen species (ROS) generation was measured using cell permeant fluorogenic dye 2′, 7′-dichlorofluorescin diacetate (DCFDA) (Abcam, United Kingdom). Briefly, the DCFDA dye loses its fluorescence upon enzymatic processing within the cell. Exposure to cellular ROS oxidizes the molecule into 2′, 7′-dichlorofluorescein which is highly fluorescent. 1 × 106 conidia/ml were grown in PDB for 2 h, conidia were then washed by centrifugation in 1× Buffer provided with the kit. DCFDA (40 μM) and NA or formaldehyde were added. As a positive control, conidia were treated with Tert-Butyl Hydroperoxide (TBHP) at final concentration of 0.1 mM. Conidia were incubated for 30 min in complete darkness at 37°C. Fluorescence was measured (Ex/Em = 485/535 nm) using Spark 10 M plate reader of Tecan (Männedorf, Switzerland).
2 7 dichlorofluorescin diacetate dcfda
Manufactured by Abcam
Sourced in United Kingdom, United States
2',7'-dichlorofluorescin diacetate (DCFDA) is a fluorogenic dye that is used to detect reactive oxygen species (ROS) in cells. It is commonly used as a cell-permeable indicator for oxidative stress. Once inside the cell, DCFDA is deacetylated by cellular esterases to the non-fluorescent 2',7'-dichlorofluorescin (DCFH), which is then oxidized by ROS into the highly fluorescent 2',7'-dichlorofluorescein (DCF).
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Lab products found in correlation
Lsrii cytometer, by BD (2 mentions)
Matlab, by MathWorks (2 mentions)
Pplcγ2, by BD (2 mentions)
Pacific blue conjugated anti cd3 clone ucht1, by BD (2 mentions)
Percpcy5.5 conjugated anti cd20 clone h1, by BD (2 mentions)
Pe cy7 conjugated anti cd5 clone l17f12, by BD (2 mentions)
Plck plyn, by BD (2 mentions)
Shp 1 antibody, by Santa Cruz Biotechnology (2 mentions)
Spark 10m plate reader, by Tecan (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Hrp conjugated anti mouse igg secondary antibody, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Anti rabbit igg horseradish peroxidase hrp conjugated secondary antibody, by Merck Group (1 mentions)
Cytochrome c, by Cell Signaling Technology (1 mentions)
3 4 5 dimethylthiazol 2 yl 5 3 carboxymethoxyphenyl 2 4 sulfophenyl 2h tetrazolium, by Promega (1 mentions)
Cyclin a, by Santa Cruz Biotechnology (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay reagent, by Promega (1 mentions)
9 protocols using 2 7 dichlorofluorescin diacetate dcfda
1
Quantifying Oxidative Stress in F. oxysporum
2
Cytotoxicity Assays and Apoptosis Evaluation
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CG was dissolved in 0.05% dimethyl sulfoxide (DMSO) and used for biological assays. CellTiter 96® AQueous One Solution Cell Proliferation Assay Reagent [MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] was purchased from Promega (Madison, WI, USA), and propidium iodide (PI) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies specific to PARP, caspase-3, caspase-8, caspase-9, Bcl-2, Bcl-xL, Bax, Bid, and cytochrome c were sourced from Cell Signaling Technology (Beverly, MA, USA). Anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody and anti-mouse IgG HRP-conjugated secondary antibody were obtained from Millipore (Billerica, MA, USA). Antibodies specific to p21, p27, cyclin D1, cyclin E, cyclin A, SOD-2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazoly carbocyanine chloride) was obtained from Enzo (New York, USA), FITC-annexin V apoptosis detection kit I was obtained from BD Biosciences (San Diego, CA, USA), and 2′,7′-dichlorofluorescin diacetate (DCF-DA) was procured from Abcam (Cambridge, UK).
3
RPE Cell Culture Protocols
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Trypsin, collagenase, Dulbecco's modified eagle's medium (DMEM), DMEM/HamF12 and foetal bovine serum (FBS) were from Gibco (Paisley PA4 9RF, UK), and were used for RPE cell cultures. Phloroglucinol was purchased from Sigma‐Aldrich (St. Louis, MO, USA) and was dissolved in dimethylsulfoxide (DMSO) to prepare a stock solution at 10 mg/ml. The stock solution was diluted in culture medium to obtain final concentrations in 0.1% DMSO. atRAL (Sigma‐Aldrich) was dissolved in dimethylfomamide (DMF) and diluted in serum‐free culture medium to final concentrations in 0.1% DMF. All the operations were carried out under dim red light. H2O2 (3%, Gifrer Barbezat, Décines, France) was diluted with serum‐free culture medium to final concentrations. 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyl tetrazolium bromide (MTT) was obtained from Sigma‐Aldrich and was used at 0,5 mg/ml. 2′,7′dichloro‐fluorescin diacetate (DCFDA) was from Abcam (Cambridge, UK).
4
Multiparametric Flow Cytometry for CLL
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Antibodies with the following specificities were purchased from BD Biosciences: pPLCγ2 (Alexa Fluor 488, pY759), pSYK (PE, pY348), pLCK/pLYN (Alexa Fluor 647, pY505). Phosphatase abundance was estimated by SHP-1 antibody (Santa Cruz Biotechnology). Detection of PBMC subsets was achieved with Pacific Blue-conjugated anti-CD3 (clone UCHT1) and PerCPCy5.5 conjugated anti-CD20 (clone H1), and CLL circulating tumor was confirmed for CD5 positivity with PE-Cy7-conjugated anti-CD5 (clone L17F12, all BD Biosciences). Intracellular reactive oxygen species was measured under resting and H2O2-stimulated conditions using 2′,7’-dichlorofluorescin diacetate (DCFDA) (Abcam). For assessment of intracellular species, cells were permeabilized following fixation with PFA with cold methanol at −20°C for 10 minutes. Intracellular staining was carried out at 4°C in PBS + 2% FBS. Cells were washed twice with PBS + 2% FBS and analyzed on an LSRII cytometer (BD Biosciences). Analysis was carried out using FlowJo software (TreeStar), Prism (Graphpad), and with custom tools written in R statistical computing language (R project) and MATLAB (Mathworks). Code is available upon request.
5
ROS Quantification in Adherent and Spheroid Cells
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Adherent cells were seeded at 1.5 × 104 cells/well for 48 h in flat glass-bottom 96-well plates (Corning). Spheroids were formed in ultra-low adherent plates for 48 h and transferred to flat 96-well plates and washed with 0.25 mM sodium phosphate solution. After staining with 25 µM 2′7′-dichlorofluorescin diacetate (DCFDA) (Abcam) in Krebs–Ringer phosphate buffer for 45 min at 37 °C, internal ROS was quantified using a TECAN plate reader (485 ex/535 em). ROS production was normalized to total protein using BCA analysis.
6
Intracellular ROS Levels Quantification
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ROS levels were detected by 2′,7′-dichlorofluorescin diacetate (DCFDA; Abcam, Cambridge, MA, USA), which is oxidized by intracellular ROS to 2′,7′-dichlorofluorescein (DCF), a highly fluorescent compound. Briefly, 10 μL of cell homogenates of each treatment group was mixed with 90 μL of phosphate buffer (pH 7.4) in black 96-well plates and treated with 50 μM DCFDA per well. Fluorescence intensity was measured by a microplate reader (Molecular Devices Corp., Sunnyvale, CA, USA) using an excitation wavelength at 485 nm and emission wavelength at 535 nm.
7
Measuring Intracellular and Extracellular ROS
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MOSE cells were seeded at 2.5 x 104 cells/well and incubated in normoxic and hypoxic conditions for 24 h in flat, glass bottom 96-well microplates. The cells were washed with 0.25mM sodium phosphate solution (pH to 7.4) warmed to 37°C 30 min prior to the experiment. Cells were stained with 25µM 2’7’-dichlorofluorescin diacetate (DCFDA) (Abcam) in Krebs-ringer phosphate buffer for 45 min at 37°C to measure H2O2 levels in live cells as previously described (36 (link), 37 (link)). ROS production was quantified using a TECAN plate reader measuring the excitation fluorescence (set at 485ex/535em). MOSE-E cells treated with 1mM H2O2 served as positive control. To measure extracellular ROS production, we used Amplex Red (ThermoFisher Scientific) at 50µM in combination with 10U/ml horseradish peroxidase, according to the manufacturer’s instructions; fluorescence was read at 571ex/585em. ROS production was normalized by the protein concentration.
8
Multiparametric Flow Cytometry for CLL
9
DCFDA Assay for Intracellular ROS
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The DCFDA-Cellular Reactive Oxygen Species Detection Assay using the cell-permeable reagent 2′,7′-dichlorofluorescin diacetate (DCFDA) (Abcam, Tokyo, Japan) to measure intracellular hydroxyl, peroxyl and other ROS activity was performed according to the assay protocol. Briefly, HSCsCygb-WT and HSCsCygb-TG were induced to produce ROS by different doses of H2O2 (0.625 to 40 µM) administrated on day 2 of cell culture. After 48 h of H2O2 treatment, both the stimulated and non-treated cells were washed twice with PBS and incubated with a working concentration of DCFDA (25 mM) for 30 min at 37 °C. After diffusion into the cell, DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2′, 7′-dichlorofluorescein (DCF). DCF is a highly fluorescent compound that can be detected by fluorescence spectroscopy with maximum excitation and emission spectra of 495 nm and 529 nm, respectively.
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