Dichlorofluorescin

ChemicalsRhodamine 123, collagenase, bovine serum albumin (BSA), N-(2-hydroxyethyl) piperazine-N’-(2-ethanesulfonic acid) (HEPES), reduced and oxidized glutathione (GSH and GSSG), acridine orange, 2’,7’-dichlorofluorescin diacetate (DCFH-DA), trichloroacetic acid, trypan blue, heparin and diclofenac sodium were purchased from Sigma-Aldrich Co. (Taufkirchen, Germany). All other chemicals were of the highest commercial grade available.
AnimalsMale Sprague-Dawley rats weighing 280 to 300 g were housed in ventilated plastic cages over PWI 8-16 hardwood bedding. There were 12 air changes per hour, 12 h light photoperiod (lights on at 08:00 h) and an environmental temperature of 21-23°C with a 50-60% relative humidity. The animals were fed with a normal standard chow diet and tap water ad libitum. All experiments were conducted according to the ethical standards and protocols approved by the Committee of Animal Experimentation of Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Isolation and incubation of hepatocytesHepatocytes were obtained by collagenase perfusion of the liver and the viability was assessed by plasma membrane disruption determined by trypan blue (0.2 w/v) exclusion test (19 (link)). Cells were suspended at a density of 10⁶ Cells/mL in round-bottomed flasks rotating in a water bath maintained at 37°C in Krebs-Henseleit buffer (pH = 7.4), supplemented with 12.5 mM HEPES under an atmosphere of 10% O₂, 85% N₂ and 5% CO₂. Each flask contained 10 mL of hepatocyte suspension. Hepatocytes were preincubated for 30 min prior to addition of chemicals. Stock solutions of all chemicals (×100 concentrated for the water solutions or ×1000 concentrated for the methanolic solutions) were prepared fresh prior to use. To avoid either non-toxic or very toxic conditions in this study, we used EC₅₀ concentrations for diclofenac in the isolated hepatocytes. The EC₅₀ of a chemical in hepatocyte cytotoxicity assessment technique (with the total 3 h incubation period) is defined as the concentration, which decreases the hepatocyte viability down to 50% following the 2 h of incubation (20 (link)). In order to determine this value for diclofenac, dose-response curves were plotted and then EC₅₀ was determined based on a regression plot of three different concentrations (data and curves are not shown). To incubate diclofenac which is soluble in methanol, with the required concentration, we prepared methanolic stock solution (×1000 concentrated) and to achieve the required concentration in the hepatocytes, we added 10 μL samples of the stock solution to the 10 mL cell suspension. Ten μL of methanol did not affect the hepatocyte viability after 3 h incubation (data are not shown)
Cell viabilityThe viability of isolated hepatocytes was assessed from the intactness of the plasma membrane as determined by the trypan blue (0.2% w/v) exclusion test (19 (link)). Aliquots of the hepatocyte incubate were taken at different time points during the 3-h incubation period. At least, 80-90% of the control cells were still viable after 3 h.
Determination of reactive oxygen speciesTo determine the rate of hepatocyte reactive oxygen species (ROS) generation induced by diclofenac, dichlorofluorescein diacetate (DCFH-DA, 1.6 μM) was added to the hepatocytes. It penetrates hepatocyte cells and becomes hydrolyzed to non-fluorescent dichlorofluorescein (DCFH). The latter then reacts with ROS to form the highly fluorescent dichlorofluorescein (DCF), which effluxes the cell. The fluorescence intensity of DCF was measured using a Shimadzu RF5000U fluorescence spectrophotometer. Excitation and emission wavelengths were 500 nm and 520 nm, respectively. The results were expressed as fluorescent intensity per 10⁶ cells (21 ).
Lipid peroxidation assayHepatocyte lipid peroxidation was determined by measuring the amount of thiobarbituric acid reactive substances (TBARS) formed during the decomposition of lipid hydroperoxides by following the absorbance at 532 nm in a Beckman DU-7 spectrophotometer (22 (link)).
Intracellular GSH and extra cellular GSSG assessmentGSH and GSSG were determined according to the spectrofluorometric method (23 (link)). Each sample was measured in quarts cuvettes using a fluorimeter set for 350 nm excitation and 420 nm emission wavelengths.
Mitochondrial membrane potential assayMitochondrial uptake of the cationic fluorescent dye, rhodamine123 (1.5 μM), has been used for the estimation of mitochondrial membrane potential. The amount of rhodamine123 remaining in the incubation medium was measured fluorimetrically using a Shimadzu RF5000U fluorescence spectrophotometer set at 490 nm excitation and 520 nm emission wavelengths. The capacity of mitochondria to take up the rhodamine123 was calculated as the difference (between control and treated cells) in rhodamine123 fluorescence. Our data were shown as the percentage of mitochondrial membrane potential collapse (%ΔΨm) in all treated (test) hepatocyte groups (24 (link)).
Lysosomal membrane integrity assayHepatocyte lysosomal membrane stability was determined from the redistribution of the fluorescent dye, acridine orange. Aliquots of the cell suspension (0.5 mL) that were previously stained with acridine orange (5 μM) were separated from the incubation medium by 1 min centrifugation at 1000 rpm. The cell pellet was then resuspended in 2 mL of fresh incubation medium. This washing process was carried out twice to remove the fluorescent dye from the media. Acridine orange redistribution in the cell suspension was then measured fluorimetrically using a Shimadzu RF5000U fluorescence spectrophotometer set at 495 nm excitation and 530 nm emission wavelengths. Lysosomal membrane damage was determined as the difference in redistribution of acridine orange from lysosomes into cytosol between treated cells and control cells at the time of preparation. Our data were shown as the percentage of lysosomal membrane leakiness in all treated (test) hepatocyte groups (25 (link)).
Determination of proteolysisProteolysis was monitored using a fluorescence assay for tyrosine release (adapted from (26 (link))). An aliquot of the hepatocyte suspension was precipitated with an equal volume of 20% trichloroacetic acid and allowed to stand overnight at 4°C. The sample was vortexed and centrifuged in a benchtop clinical centrifuge (at 17,320×g) for 15 min. A volume of 1 mL aliquot of supernatant was removed and placed in a test tube to which 1 mL of 0.2% solution of 1-nitroso-2-naphthol and 1macid nitrite reagent (10 mg/mL NaNO2 in 20% HNO3) was added. The solution was vortexed, covered with parafilm and incubated at 37°C for 30 min.
A volume of 5 mL ethylene dichloride was added to the test tube, the mixture was vortexed vigorously and the sample was centrifuged for 10 min at high speed. The fluorescence of the aqueous phase was read in a Shimadzu RF5000U spectrophotometer (excitation at 460 nm and emission at 570 nm). The tyrosine content of the sample was determined from a standard curve constructed from known concentrations of tyrosine (0-100 μM).
Determination of caspase-3 activityCaspase-3 activity was determined in cell lysate of hepatocytes from different treatments using “Sigma’s caspase-3 assay kit (CASP-3-C)” (27 (link)). In brief, this colorimetric assay is based on the hydrolysis of substrate peptide, Ac-DEVD-pNA, through caspase-3. The released moiety (p-nitroaniline) has a high absorbance at 405 nm. The concentration of the p-nitroaniline (μM) released from the substrate is calculated from the absorbance values at 405 nm or from a calibration curve prepared with defined p-nitroaniline solutions.
Statistical analysisLevene’s test was used to check the homogeneity of variances. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s HSD as the post-hoc test. Results were presented as mean ± SD of triplicate samples. The minimal level of significance chosen was p < 0.05.

To determine intracellular ROS production, differentiated C2C12 myotubes were incubated with the fluorogenic dye 2′,7′‐dichlorofluorescin diacetate (DCF‐DA) (D6883; Sigma‐Aldrich), which is oxidized by ROS to produce DCF and emits green fluorescence from the dye (Kim & Xue, 2020 (link)). Cells were plated at a density of 1.3 × 10⁵ cells per well in a 24‐well plate and randomly divided into the following groups: the Veh, AII, AII + 100 nM VD₃, AII + 10 nM 1,25VD₃, and AII + Lo groups. All treatments were applied at the differentiation phase as follows: 1 μM AII at Days 4–6, 100 nM VD₃ and 10 nM 1,25VD₃ at Days 5–6 and 10 μM losartan applied 2 h before AII was administered on Day 4. Myotubes incubated overnight with 20 μM hydrogen peroxide (H₂O₂) (88597; Millipore, Temecula, CA, USA) were used as a positive control. On Day 7, DCF‐DA staining was performed according to a previous report (Kim & Xue, 2020 (link)) with slight modifications and all procedures were protected from light. After the culture medium was removed and washed once with warm phenol red‐free DMEM (17‐205‐CV; Corning), the myotubes were incubated with 10 μM DCF‐DA solubilized with methanol in phenol red‐free DMEM at 37°C in a 5% CO₂ incubator for 30 min in the dark. The medium containing DCF‐DA was then removed, and the cells were washed once with phenol red‐free DMEM and twice with PBS. Representative fluorescence images using the FITC channel were captured with an Olympus Inverted Fluorescence Microscope Model IX83 (Olympus, Tokyo, Japan) equipped with an ORCA‐Flash 2.8 Digital CMOS Camera (C11440) (Hamamatsu Photonics, Hamamatsu, Japan) at ×100 magnification.
To measure fluorescence intensity using a microplate reader, protein was extracted from treated myotubes using ice‐cold RIPA buffer containing a cocktail of protease inhibitors (P8340; Sigma‐Aldrich). The extracted proteins were centrifuged at 21,130 g for 10 min (4°C) and 100 μL of the supernatant was transferred to a black clear bottom 96‐well plate (3603; Corning). The fluorescence intensity was measured using a multimode microplate reader (Infinite® M200 PRO; Tecan Trading AG, Männedorf, Switzerland) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. A BCA assay (Thermo Scientific) was performed to determine the protein concentration, which was used to normalize the DCF fluorescence intensity.

Mock or Infected BMDMs were seeded in a black, clear bottom 96-well microplate at a cell density of 5 × 10⁴ cells/well. The cells were assayed using the DCFDA Cellular ROS Detection Assay reagent (Sigma 4091-99-0). Cells were analyzed using fluorescence microscope and images were captured at excitation and emission wavelengths of 485 and 535 nm, respectively. The images were quantified with ImageJ using Hoechst 33342 (Thermo Scientific 62249) nuclear stain to count the total number of cells.