Characteristics of MSC, intracellular reactive oxygen species (ROS) production, and the CD11b expression of PMN were determined by FACS. Briefly, the prepared cell suspensions of MSC were incubated with specific antibodies using phycoerythrin (PE)-anti-CD34, PE-anti-CD45, fluorescein isothiocyanate (FITC)-anti-CD29, and FITC-anti-CD44 (e-BIOSCIENCE, USA) before measurement of PMN characteristics. And the mixture of blood or BALF with LDS-751 was incubated with saturating concentrations of FITC-labeled mouse CD11b antibody (Sigma, USA), and 80 mmol/L 2’,7’-dichlorofluorescin diacetate (Molecular Probes, USA) before measurement of CD11b and intracellular ROS production of PMN. And the analysis was performed on an FACS using CellQuest software (Becton Dickinson, USA).
2 7 dichlorofluorescin diacetate
Manufactured by Thermo Fisher Scientific
Sourced in United States
2',7'-dichlorofluorescin diacetate is a fluorogenic compound used for the detection of reactive oxygen species in biological systems. It is a non-fluorescent precursor that becomes fluorescent upon oxidation.
Automatically generated - may contain errors
Lab products found in correlation
Quantichrom arginase assay kit, by BioAssay Systems (2 mentions)
Anti cd44 fitc, by Thermo Fisher Scientific (1 mentions)
Cellquest software, by BD (1 mentions)
Anti cd45 pe, by Thermo Fisher Scientific (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Abcam (1 mentions)
Spectramax gemini, by Molecular Devices (1 mentions)
Softmax pro software, by Molecular Devices (1 mentions)
Cell culture plates, by Thermo Fisher Scientific (1 mentions)
Tryptophan, by Merck Group (1 mentions)
Phenylalanine, by Merck Group (1 mentions)
Valine, by Merck Group (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Trypan blue, by Merck Group (1 mentions)
L tryptophan, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
L phenylalanine, by Merck Group (1 mentions)
L valine, by Merck Group (1 mentions)
Congo red, by Merck Group (1 mentions)
Sodium azide, by Merck Group (1 mentions)
17 protocols using 2 7 dichlorofluorescin diacetate
1
Characterization of MSC and PMN
2
Evaluating NET Formation and ROS in Neutrophils
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The formation of NETs was evaluated at basal state and at activated state using the membrane impermeable DNA-binding dye, SYTOX green (Molecular Probes, Invitrogen Life Technologies). Experiments were performed in 96-well culture plates. Freshly isolated neutrophils (2 × 105/well) were stimulated with a protein kinase C activator, phorbol 12-myristate 13-acetate (PMA, 0.01 μg/mL final; Biovision, USA) for 1 h. Following PMA stimulation or basal state, cells were incubated with 2 μM SYTOX green to detect extracellular DNA. After 30 min, neutrophils were washed and resuspended in RPMI and the plates were read using the SpectraMAX Gemini fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA) at excitation and emission wavelengths of 500 nm and 530 nm, respectively. The data were analyzed using SoftMax Pro Software (Molecular Devices). To measure ROS, isolated neutrophils were incubated at 37°C for 30 min in the presence of 2 μM 2′,7′-dichlorofluorescin diacetate (Molecular Probes), which is used to detect hydrogen peroxide, peroxyl radicals, and peroxynitrite anions. To evaluate NET formation following 1 h PMA stimulation, extracellular fluorescence was measured using a spectrofluorometer at excitation and emission wavelengths of 488 nm and 527 nm, respectively.
3
Quantifying Neutrophil H2O2 Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure H2O2 production, isolated neutrophils or PBMCs (4 x 106 cells/ml) were incubated with 2’,7’-dichlorofluorescin diacetate (final concentration 0.5 mM; Molecular Probes/Invitrogen, Carlsbad, CA, USA) for 10 min at 37°C. Fifty microliters of cell suspension was sampled before, and 10 min after, the addition of 50 ml of opsonized, propidium iodide-labeled Staphylococcus aureus (final concentration ~108 bacteria/ml). Samples were analyzed for uptake of labeled bacteria and oxidation of 2’,7’-dichlorofluorescin diacetate to fluorescent 2’,7’-dichlorofluorescein by flow cytometry as previously described (57 (link), 76 (link)). The LDN population in the PBMC fraction was defined for analysis by gating on CD66b+ cells.
4
Amino Acid-coated Gold Nanoparticles and PC12 Cell Viability
Check if the same lab product or an alternative is used in the 5 most similar protocols
Cell culture medium, RPMI 1640 (Caisson Laboratories, Logan, UT, USA), fetal bovine serum (Caisson Laboratories, Logan, UT, USA), horse serum (Gibco Company, Grand Island, NY, USA), Solutions streptomycin (CMG Company, Isfahan, Iran), penicillin and Trypsin / EDTA (Invitrogen, Paisley, Scotland) , Cell culture plates (Nunc, Roskilde, Denmark), Tetrachloroauric acid (Sigma, MO, USA), L-aspartic acid (Sigma, MO, USA), L- glutamic acid (Sigma, MO, USA), L- tryptophan (Sigma, MO, USA), L- phenylalanine (Sigma, MO, USA), L- valine (Sigma, MO, USA), Trisodium citrate (Sigma, MO, USA), Trypan blue (Sigma, MO, USA), Sodium azide (Sigma, USA), Congo red (Sigma, MO, USA), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-MTT (Sigma, Germany), Acridine orange (AO) and Ethidium bromide (EtBr) (AB, Uppsala, Sweden), PC12 cell line from the Pasteur Institute of Iran, 2′,7′-Dichlorofluorescin diacetate from Molecular Probes (Eugene, OR, USA), Dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany).
In this empirical study, the effect of five amino acids contains , phenylalanine-coated, tryptophan-coated, glutamate-coated, aspartate-coated and valine-coated gold nanoparticles on type and death rate of PC12 cancer cells was investigated.
+ Expand
Cell culture medium, RPMI 1640 (Caisson Laboratories, Logan, UT, USA), fetal bovine serum (Caisson Laboratories, Logan, UT, USA), horse serum (Gibco Company, Grand Island, NY, USA), Solutions streptomycin (CMG Company, Isfahan, Iran), penicillin and Trypsin / EDTA (Invitrogen, Paisley, Scotland) , Cell culture plates (Nunc, Roskilde, Denmark), Tetrachloroauric acid (Sigma, MO, USA), L-aspartic acid (Sigma, MO, USA), L- glutamic acid (Sigma, MO, USA), L- tryptophan (Sigma, MO, USA), L- phenylalanine (Sigma, MO, USA), L- valine (Sigma, MO, USA), Trisodium citrate (Sigma, MO, USA), Trypan blue (Sigma, MO, USA), Sodium azide (Sigma, USA), Congo red (Sigma, MO, USA), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-MTT (Sigma, Germany), Acridine orange (AO) and Ethidium bromide (EtBr) (AB, Uppsala, Sweden), PC12 cell line from the Pasteur Institute of Iran, 2′,7′-Dichlorofluorescin diacetate from Molecular Probes (Eugene, OR, USA), Dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany).
In this empirical study, the effect of five amino acids contains , phenylalanine-coated, tryptophan-coated, glutamate-coated, aspartate-coated and valine-coated gold nanoparticles on type and death rate of PC12 cancer cells was investigated.
5
Quantifying Oxidative Stress in Sperm
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dihydroethidine (DHE; Molecular Probes, Inc.) and 2′,7′-dichlorofluorescin diacetate (DCFH-DA; Molecular Probes, Inc.) detected superoxide anion ( ) and H2O2, respectively. DCFH-DA is a stable compound that passively diffuses into cells and is hydrolyzed by intracellular esterase to yield DCHF, which is trapped inside cells. H2O2 produced by cells oxidizes DCFH to the highly fluorescent compound, 2,7-dichlorofluorescein (DCF) which is fluorescent at 530 nm. The DHE can be directly oxidized into ethidium bromide by produced by sperm. We modified the method used by Fisher et al [17 (link)] to evaluate H2O2 levels in sperm; sperm levels were measured using a modification of a previously described method [18 (link)]. DCFH-DA (2.5 mM) and DHE (0.33 mM), were added to the sperm suspension at a concentration of 1–2 × 106 sperm/mL and incubated at 34°C for 30 minutes for both DCFH-DA and DHE. The final concentrations of DCHF-DA and DHE were 12.5 μM and 1.65 μM, respectively. Each aliquot was analyzed using FACScan FCM (Becton Dickinson FACScan). Intracellular H2O2 and levels were measured with excitation and emission settings at 488 nm and 530 nm, respectively. Data were expressed as the percentage of fluorescent spermatozoa which indicated positive ROS cells [19 (link)].
6
HDL Antioxidant and PON1 Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HDL was prepared by the dextran-Mg 2+ method [21] (link). The antioxidant properties of HDL were tested in the presence or absence of oxidized LDL as described [22] with some modifications [23] . In short, 2,7-dichlorofluorescin diacetate (Molecular Probes/Invitrogen, Carlsbad, CA, USA) was dissolved in fresh methanol at 2.0 mg/mL and incubated at room PON1 activity in HDL, prepared by the dextran-Mg 2+ method, was determined using paraoxon as substrate [24] . Briefly, the assays were performed in a final volume of 250 µL PrePrint (Unreviewed) Full peer-reviewed manuscript: doi:10.1016/j.bbalip.2015.09.006. containing 5µL of dextran-Mg 2+ -prepared HDL, 5.5 mmol/L paraoxon (paraoxon-ethyl, Sigma Aldrich), 2 mmol/L CaCl2 and 100 mmol/L Tris-HCl, pH 8.0. The rate of pnitrophenol formed by hydrolysis of paraoxon was measured by monitoring the increase in absorbance at 405nm for 25min at room temperature in a microplate spectrophotometer.
PON1 activity was expressed as U per L. 1U is defined as the activity that catalyzes the formation of 1 μmol p-nitrophenol per min.
PON1 activity was expressed as U per L. 1U is defined as the activity that catalyzes the formation of 1 μmol p-nitrophenol per min.
7
Measuring Oxidative Stress in Mouse Mesangial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary mouse mesangial cells (pMMCs) from wild-type and Nox knockout mice were serum starved for 6 h and then stimulated with recombinant human TGF-β1 (10 ng/ml) for 10 min. The cells were washed with Hank's balanced salt solution (HBSS) and incubated for 10 min in the dark at 37 °C in HBSS containing 10 μM DCF-DA (2',7'-dichlorofluorescindiacetate, Molecular Probes). Fluorescence from oxidized DCF was detected by a Zeiss LSM510 confocal microscope (version 2.3 Minneapolis, MN, USA) at excitation and emission wavelengths of 488 nm and 515-540 nm, respectively. Five fields were randomly selected for fluorescence measurements and the mean relative fluorescence intensity was used. All the experiments were repeated at least three times.
8
Oxidative Stress Response to Oxygen Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were stained with 5 µM cell-permeant 2′,7′-dichlorofluorescin diacetate, an oxidative stress indicator (Invitrogen; Thermo Fisher Scientific, Inc.), for 15 min at 37°C, washed in PBS, trypsinized and resuspended at 1×106 cells/ml. The dichlorofluorescein signal was observed and analyzed with a FACSCalibur flow cytometer and CELLQuest software version 3.3 (BD Biosciences, Franklin Lakes, NJ, USA). Cells were pre-treated with 10 µM CGRP for 24 h, and incubated with air or 60% oxygen to examine its effects on ROS production. Cells were maintained at 37°C for 24 h and dichlorofluorescein was examined in a 5% oxygen tissue culture incubator to determine the effect of reduced ambient oxygen on ROS levels.
9
Intracellular ROS Measurement in F. solani
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The intracellular ROS in F. solani was measured using 2′,7′-dichlorofluorescin diacetate (Invitrogen) as described (30 (link)). F. solani (106 conidia/ml) were incubated with 2′,7′-dichlorofluorescin diacetate in the presence or the absence of S100A12 (25 μM) for 2 h, washed with 1× PBS, and checked by fluorescence microscope (Axio Vert.A1; Zeiss) using 20× objective or analyzed by flow cytometry (Beckman Coulter). The mean fluorescence intensity was determined from two separate experiments. ROS generation in HCEC was also determined in a similar manner.
10
ROS Measurement in Aβ-Treated BV2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BV2 cells were harvested in a FACS tube after treatment with Aβ (1~4 µM) and TDCA (400 ng/ml) for 24 h. Cells were washed with DPBS and incubated with 5 μM 2′-7′-dichlorofluorescin diacetate (Invitrogen, Eugene, Oregon, US) in DMEM containing 1% penicillin and streptomycin for 30 min at room temperature in the dark. Samples were washed in DPBS, suspended in FACS buffer containing DAPI (0.3 µg/ml), immediately acquired and analyzed using a BD LSR Fortessa flow cytometer (BD Bioscience, San Jose, CA, USA) and FlowJo v9 software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!