To measure ROS levels, neutrophils were labeled with 5 μM dihydrorhodamine (Sigma-Aldrich) and incubated with or without PMA at 37 °C for 30 minutes. To assess CD11b expression and platelet-neutrophil aggregates, leukocytes in PBS-diluted blood were labeled with fluorescein isothiocyanate-conjugated anti-CD45 and phycoerythin-conjugated anti-CD11b or -CD41 antibodies (Biolegend San Diego, CA, USA), or corresponding isotype controls, during 15 min. with or without 50 nM PMA. Cells were analyzed in a flow cytometer. The neutrophil population was selected based on CD45 expression and side scatter (SSC) and platelet-neutrophil aggregates were identified as the percentage of events staining positive for CD41. Baseline CD11b was calculated as the ratio between mean fluorescence intensity (MFI) of antibody staining and the isotypic control. Fold-increase in CD11b was defined as the ratio between MFI of the stimulated sample minus baseline CD11b and baseline CD11b.
Dihydrorhodamine
Manufactured by Merck Group
Sourced in United States
Dihydrorhodamine is a fluorogenic probe that can be used to detect reactive oxygen species (ROS) in biological systems. It is non-fluorescent in its reduced state but becomes highly fluorescent upon oxidation, allowing for the visualization and quantification of ROS levels in cells and tissues.
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Lab products found in correlation
Facscan, by BD (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Reduced glutathione, by Merck Group (1 mentions)
Sephadex g 50, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Ubiquitin, by Merck Group (1 mentions)
5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (1 mentions)
2 4 dinitrophenylhydrazine, by Merck Group (1 mentions)
Chymotrypsinogen, by Merck Group (1 mentions)
Hoescht 33342 dye, by Merck Group (1 mentions)
Hemoglobin, by Merck Group (1 mentions)
Ethoxyresorufin, by Merck Group (1 mentions)
Tyrosin, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Myoglobin, by Merck Group (1 mentions)
N zno, by Merck Group (1 mentions)
Xfe24 extracellular flux analyzer, by Agilent Technologies (1 mentions)
Mito stress test kit, by Agilent Technologies (1 mentions)
11 protocols using dihydrorhodamine
1
Quantifying Neutrophil Activation and Aggregation
2
Zinc Oxide Nanoparticle Assays
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Suspensions of n-ZnO (mean particle size 35 nm), reduced glutathione (GSH), bovine serum albumin, phenylmethylsulfonyl fluoride (PMSF), 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), nicotinamide adenine dinucleotides (NADH, NAD, NADPH), EDTA, dihydrorhodamine, Hoescht 33342 dye, 2,4-Dinitrophenylhydrazine, tyrosin, hemoglobin, chymotrypsinogen, cytohrome c, myoglobin, ubiquitin, Sephadex G-50, β-mercaptoethanol, ethoxyresorufin, certified reference material ERM-BB422 and Lactobacillus leichmannii D-Lactate dehydrogenase were purchased from Sigma Chem. Co. (St. Louis, USA). All other chemicals were obtained from the Synbias (Kyiv, Ukraine), Bayer (Kyiv, Ukraine) and Balkanpharma-Dupnitsa (Dupnitsa, Bulgaria) commercial suppliers. All reagents were of the analytical grade or higher.
3
Measurement of Macrophage Respiratory Burst
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The respiratory burst indicated by proton production rate (PPR) was measured as previously described (Liu et al., 2013a (link)). Briefly, macrophages were sorted from peritoneal cells and incubated with dihydrorhodamine (1 M, Sigma-Aldrich-P8139, St.Louis, MO, USA). Samples were incubated at 37°C for 15 min and subjected to flow cytometry analysis. Oxygen consumption rate (OCR) was measured with an XFe24 extracellular flux analyzer (Seahorse Bioscience) according to the manufacturer's instructions as described previously (Lu et al., 2018 (link)). In brief, macrophages sorted from peritoneal exudate cells activated with lipopolysaccharide (LPS) for 24 h, or IL-4 for 48 h were seeded in XFe24 microplates (5 × 105) to immobilize the cells. Wash the cells with XF base medium with glucose (10 mM), sodium pyruvate (1 mM), L-glutamine (2 mM) (referred to as the assay medium). After incubation in the assay medium in an incubator without CO2 for 1 h, cells were subjected to oxygen consumption assays with a Mito stress test kit (Seahorse Biosciences). oligomycin (1 μM), FCCP (1 μM), rotenone/antimycin A (0.5 μM) were added into medium subsequence. The data were acquired on the Seahorse XF-24 and analyzed on the Wave.
4
Dihydrorhodamine Staining of Endothelial Cells
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ECs were washed with PBS and incubated in 20 μmol/L of dihydrorhodamine (Sigma) with 0.5% FBS for 30 min. At the end of incubation, the buffer was aspirated and the cells were washed with 1 × PBS and then were imaged.
5
Measuring ROS Production in Mesangial Cells
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ROS production was measured with dihydro-rhodamine as previously described 29 (link). Briefly, 1.5 × 105 mesangial cells were plated in six-well plates in Dulbecco's modified Eagle's medium (DMEM) containing 1% fetal calf serum (FCS). After 2 days, the cells were washed with PBS and then incubated in serum-free medium with 2 μM dihydro-rhodamine (Sigma) for additional 2 hours. The cells were then trypsinized and the generation of fluorescent rhodamine 123 was analyzed by a FACScan (BD Bioscience). Three independent experiments were performed in triplicate.
6
Neutrophil Activation Assay in Cows
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Neutrophils (3 × 106 cells/mL final concentration, 1 mL/well final volume) from cows in early (n = 9) and mid-lactation (n = 9) were resuspended in media 199 containing 10% FBS, assigned one of three doses of choline, and cultured for 2 h at 37°C and 5% CO2. Due to methodological errors, samples from two cows were excluded from this analysis. Next, cells were primed with or without LPS (1 μg/mL, final concentration, Escherichia coli O55:B5, Sigma-Aldrich Co.) for 30 min followed by the addition of dihydrorhodamine (100 μM, final concentration, Sigma-Aldrich). Ten minutes after incubation with dihydrorhodamine, cells were challenged with E. coli covalently labeled with Texas Red® (Molecular Probes Inc.) at a ratio of 20:1 (bacteria:neutrophil) and returned to incubation for 40 min. After incubation, neutrophil activity was stopped by cold shock. Lastly, the cells were washed and pelleted three times and finally reconstituted in 200 μL of phosphate-buffered saline for fluorescence measurement.
7
Quantifying Intracellular ROS Levels
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The oxidant-sensitive fluorescent probe dihydrorhodamine (DHR; Sigma-Aldrich, St. Louis, MO, USA) was used to determine the intracellular ROS levels. Cells were treated with 50 µM DHR in culture media for 1 h, and then washed with 0.9% NaCl twice and suspended in 200 µL of 0.9% NaCl. Cells treated with 20 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) during 10 min before incubation with DHR were used as positive control. Fluorescence was measured in FL-1 channel (533/530 nm) in a BD Accuri™ C6 plus Personal Flow Cytometer. Data was acquired from a total of 20,000 events/samples from 2 independent assays. Quantification of intracellular ROS was expressed by relative fluorescence units (RFU) calculated in relation to sample control (unlabeled cells).
8
Monocyte Oxidative Stress Measurement
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Monocytes were seeded into 6-well plates at a density of 106 cells/well in medium with 10% human serum and incubated until semi-confluence. The medium was then replaced by CM in treated wells or by the medium in controls. After 24 h incubation, cells were stimulated with 1 μg/ml of LPS (Sigma-Aldrich) for 30 min. After washing with medium without phenol red, a solution of dihydrorhodamine (5 μM, Sigma-Aldrich) in this medium was added and cells were incubated for 15 min at 37°C. The supernatant was then discarded, cells were washed several times with PBS and resuspended in PBS to measure the fluorescence (excitation 485 nm/emission 534 nm) in a FACS-Canto II (BD Biosciences) flow cytometer.
9
Neutrophil Oxidative Burst Assay
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Isolated bone marrow neutrophils were incubated in the presence of 1 μM dihydrorhodamine (Sigma) during stimulation with different concentrations of PMA (Sigma) for 15 minutes or LPS for 4 hours (Sigma). The oxidative burst of neutrophils was then analyzed by flow cytometry.
10
Antioxidative Capacity of ApoB-Depleted Serum
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As described [29 (link)], the antioxidative capacity of apoB-depleted serum was determined with a fluorometric assay by using the fluorescent dye dihydrorhodamine (D1054, Sigma-Aldrich, Darmstadt, Germany). The dye was dissolved in DMSO (50 mM stock), diluted in HEPES (20 mM HEPES, 150 mM NaCl2, pH 7.4) containing 1 mM 2,2′-azobis-2-methyl-propanimidamide-dihydrochloride (440914, Sigma-Aldrich, Darmstadt, Germany) to yield a 10 μM working reagent. Into a 384-well plate, 10 µL apoB-depleted serum dilution (1:10) was added, and 90 µL of working reagent was added. The increase in fluorescence as a result of dihydrorhodamine oxidation was monitored at 538 nm for 90 min. The increase in dihydrorhodamine fluorescence per minute in absence of apoB-depleted serum was set at 100%, and the individual apoB-depleted serum samples were expressed as a percentage of inhibition of dihydrorhodamine oxidation.
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