All chemicals were obtained from Sigma Chemical (St. Louis, MO, USA) unless otherwise indicated. Cell culture reagents were purchased from Gibco BRL (Rockville, MD, USA). Antibodies against HO-1, p-Akt, Akt, AMPKα, p-AMPKα and Akt inhibitor LY294002 were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibody against each of Nrf2 and phosphatase and tensin homolog and horse-raddish peroxidase-conjugated secondary antibodies were purchased from SantaCruz Biotechnology (SantaCruz, CA, USA). The 2′,7′-dichlorofluorescin diacetate (DCF-DA) was purchased from Invitrogen (Carlsbad, CA, USA). Hank’s balanced salt solution (HBSS) was purchased from Meditech (Herndon, VA, USA).
2 7 dichlorofluorescin diacetate dcf da
Manufactured by Thermo Fisher Scientific
Sourced in United States
2',7'-dichlorofluorescin diacetate (DCF-DA) is a fluorogenic dye that is widely used for detecting reactive oxygen species (ROS) in biological systems. It is a cell-permeable compound that can be oxidized by ROS to produce the highly fluorescent compound 2',7'-dichlorofluorescein, which can be detected using fluorescence techniques.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Cell Signaling Technology (2 mentions)
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Dihydroethidium dhe, by Thermo Fisher Scientific (2 mentions)
Arl13b 17711 1 ap, by Proteintech (2 mentions)
4 6 diamidno 2 phenylinole dapi, by Merck Group (2 mentions)
Chicken anti mouse igg alexa 488 a 21200, by Thermo Fisher Scientific (2 mentions)
β tubulin t4026, by Merck Group (2 mentions)
Acetylated tubulin t7451, by Merck Group (2 mentions)
Goat anti rabbit alexa 568 a 11011, by Thermo Fisher Scientific (2 mentions)
Cell culture reagents, by Thermo Fisher Scientific (1 mentions)
P ampkα, by Cell Signaling Technology (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Antibodies against ho 1, by Cell Signaling Technology (1 mentions)
Diphenyleneiodonium dpi, by Merck Group (1 mentions)
Cytomics fc500, by Beckman Coulter (1 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Lysotracker green, by Thermo Fisher Scientific (1 mentions)
Cyto id, by Enzo Life Sciences (1 mentions)
Trypsin edta, by PanEco (1 mentions)
Mitotracker green mtg, by Thermo Fisher Scientific (1 mentions)
26 protocols using 2 7 dichlorofluorescin diacetate dcf da
1
Antioxidant Signaling Pathway Analysis
2
Quantifying Oxidative Stress in hMSC Attachment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate the oxidative stress generated while the cells attach to the titanium surface, we placed a total of 1 × 104 hMSC cells in 100 µL on the surface of each sample in a 12-well plate, immediately and 28 days after UV treatment, with and without dH2O. After we had incubated the cells on the treated titanium disc surfaces for 24 h at 37 °C in 5% CO2, we detected the generation of intracellular ROS levels using 2′, 7′-dichlorofluorescin diacetate (DCF-DA; Invitrogen, Carlsbad, CA) fluorophotometry. We incubated the cells with 100-µM DCF-DA for 1 h at 37 °C in 5% CO2. The peak excitation wavelength for the oxidised DCF was 492 nm, while for the emission, it was 527 nm. We used confocal laser-scanning microscopy to observe the intracellular ROS signal, while the fluorescence density was quantitatively determined using the ImageJ software (NIH, Bethesda, MD, USA).
3
ROS Analysis of Activin A Chondrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For ROS analysis, chondrocytes isolated from postnatal WT and activin A Tg mice, infected with Ad‐activin A, Ad‐Nox4, or Ad‐C, or transfected with Nox4 siRNA and then infected with the indicated MOIs of Ad‐activin A were used. DPI (diphenyleneiodonium; Sigma‐Aldrich, St. Louis, MO, USA) was added 1 h prior to infection as a positive control. To detect intracellular ROS, chondrocytes were loaded with 20 × 10−6m of 2’,7’‐dichlorofluorescin diacetate (DCF‐DA; Invitrogen, Waltham, MA, USA) and incubated for 30 min at 37 °C in the dark. After being washed and harvested, cells were immediately examined by flow cytometry using a FACS instrument (Miltenyi Biotec, Seoul, Korea) and analyzed with the FlowJo software (BD Bioscience, San Diego, CA, USA). Results are expressed as the mean fluorescence intensity. For oxidative stress analysis of mouse cartilage sections, immunohistochemical staining was performed with 8‐hydroxy‐2’‐deoxyguanosine (8‐OhdG; GTX41980, GeneTex, Irvine, CA, USA).
4
Quantifying Mitochondrial Dynamics and Autophagy in MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative measurement of mitochondrial content and mitochondrial membrane potential were performed by loading of MSC with 1 µM MitoTracker Green and MitoTracker Red (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), correspondently. To evaluate autophagy-lysosomal system, MSC were loaded with 2 µM Cyto-ID (Enzo Life Sciences, New York, NY, USA) or 1 µM LysoTracker Green (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The levels of ROS were estimated by stained with 1 µM 2′,7′-Dichlorofluorescin diacetate (DCFDA) (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Incubation cells with the probe was conducted in DMEM/F12 medium without sodium bicarbonate for 30 min at 37 °C. Then, MSC were washed with phosphate-buffered saline (PBS) and dissociated in 0.05% Trypsin-EDTA (Paneco, Moscow, Russia). The fluorescence intensity was evaluated by flow cytometry using Cytomics FC500 (Beckman Coulter, Brea, CA, USA). Cyto-ID, MitoTracker Green, LysoTracker Green and DCF-mediated fluorescence were measured on the FL1 channel, while MitoTracker Red-mediated fluorescence was evaluated on the FL3 channel. Argon laser with λex = 488 nm was used to excite the fluorescence.
5
Mitochondrial and Oxidative Stress Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Specific mitochondria sensitive dyes were reconstituted in RPMI media as follows: 50 nM Mitotracker Green (MTG; Invitrogen), 25 nM Mitotracker Deep Red (MTDR; Invitrogen), 300 nM TMRE and 5 µM 2′,7′-Dichlorofluorescin diacetate (DCFDA; Invitrogen). 100 µl each of the reconstituted dye was incubated with 5 x 105 liver non-parenchyma cells in a 96 well flat-bottom plate for 30 min (37°C, 5% CO2). After the incubation, cells were washed and stained for antigen specific CD8+ CXCR5+ T cells for flow cytometry. Data were collected and analyzed by flow cytometry as previously described.
6
Multi-Parameter Immunophenotyping of Mouse HSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Regarding the reagents used in this study, rhTPO was procured from Shenyang Sunshine Pharmaceutical Co., Ltd. (Shenyang, China). Anti-mouse CD117 (c-kit)-APC (clone2B8), anti-mouse Lineage-PercpCy5.5 (51-9006964), anti-mouse Ly-6 A/EA (Sca-1)-PE/Cy7 (cloneD7), anti-mouse CD135-PE (cloneA2F10), anti-mouse CD34-FITC (cloneRAM34), anti-mouse CD45.1-PE (cloneA20), anti-mouse CD45.2-FITC (clone104), anti-mouse CD45.1-APC, anti-mouse CD45.2-PECy7, anti-mouse B220-PE (cloneRA3-6B2), anti-mouse CD3-FITC (clone17A2), anti-mouse CD11b-PE (M1/70), anti-mouse Gr-1-FITC (cloneRB6-8C5), anti-mouse Ki67-PE (cloneSolA15), anti-mouse 7AAD-PercpCy5.5 (51-88981E), anti-mouse Annexin V-PE (640908), and anti-mouse γH2AX-FITC (clone2F3) were obtained from Biolegend (San Diego, CA, USA). Additionally, 2’, 7′-dichlorofluorescin diacetate (DCFDA) was purchased from Invitrogen (Carlsbad, CA, USA).
7
Investigating Apoptotic Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hyclone Laboratories (Logan, UT, USA) provided all cell culture reagents. Sigma-Aldrich (St. Louis, MO, USA) supplied N-acetylcysteine (NAC) and the primary antibody against β-actin. Cell Signaling Technology, Inc. (Beverly, MA, USA) supplied primary antibodies specific for cleaved caspase-9, caspase-7, caspase-3, cleaved PARP, cytochrome C, p-STAT3 (S727), p-STAT3 (Y705), STAT3, p-JAK2, JAK2, and cyclin D1. Santa Cruz Biotechnology (Dallas, TX, USA) provided antibodies against cyclin D1, D2, and D3. Novus Biologicals (Littleton, CO, USA) and Abcam (Cambridge, UK) supplied antisurvivin and anti-COX IV antibodies, respectively. Cell Signaling Technology, Inc. provided secondary antibodies conjugated with horseradish peroxidase. Invitrogen (Carlsbad, CA, USA) supplied 2',7'-dichlorofluorescin diacetate (DCF-DA). Thermo Fisher Scientific (Waltham, MA, USA) provided Hank's balanced salt solution (HBSS).
8
Sericin and Fibroin Effects on ROS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HaCaT cells were seeded in 48-well plates. We investigated if 1 h pretreatment with sericin (100 μg/ml) and I-sericin (25–100 μg/ml) or fibroin (100 μg/ml) and I-fibroin (25–100 μg/ml) affected ROS generation in cells treated with H2O2 (1 mM) for 30 min. After washing with PBS, cells were stained with 10 μM 2´7´-dichlorofluorescin diacetate (DCF-DA) (Invitrogen, Carlsbad, CA, USA) in PBS for 30 min in the dark at 37ºC. Fluorescence was measured with an excitation wavelength of 490 nm and an emission wavelength of 525 nm.
9
Neuronal Electrophysiology Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The dissecting solution used for the preparation of brain slices was composed of: sucrose (252 mM), KCl (2.5 mM), CaCl2 (0.1 mM), MgCl2 (2 mM), Glucose (10 mM), NaHCO3 (26 mM), and NaH2PO4 (1.25 mM). The extracellular fluid used for the patch-clamp recording contained: NaCl (117 mM), KCl (3.6 mM), CaCl2 (2.5 mM), MgCl2 (1.2 mM), NaH2PO4 (1.2 mM), NaHCO3 (25 mM), and glucose (11 mM). It was continually aerated with 95% O2 and 5% CO2, which maintained the pH at approximately 7.4. The pipette (internal) solution contained: K-Glu (150 mM), HEPES (10 mM), KCl (5 mM), EGTA (0.1 mM), Mg-ATP (2 mM), and NaGTP (0.3 mM). The pH was adjusted to 7.2 using KOH. In low Na+ external solution, equimolar choline-Cl replaced NaCl. The following reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA): X, XO, PBN, catalase, superoxide dismutase (SOD), 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS). Dihydroethidium (DHE) and 2′,7′-dichlorofluorescin diacetate (DCF-DA) were obtained from Molecular Probes (Eugene, OR, USA).
10
BAFF-Mediated Oxidative Stress and Apoptosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant human BAFF, biotinylated anti-human BAFF-R and TACI antibodies was purchased from R&D Systems (Minneapolis, MN, USA). Human BAFF-murine CD8 (BAFF-muCD8), biotinylated fusion proteins, were purchased from Ancell (Bayport, MN, USA). BAY61-3606, Syk inhibitor, was purchased from Adooq bioscience (Irvine, CA, USA). Catalase, dimethyl sulfoxide (DMSO), N-acetyl-L-cysteine (NAC), hydrogen peroxide (H2O2) and 3,3′-dihexyloxacarbocyanine iodide (DiOC6) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1) was obtained from Invitrogen (Eugene, Oregon, USA). 2′,7′–dichlorofluorescin diacetate (DCF-DA) was purchased from Molecular Probe (Eugene, Oregon, USA). Ac-DEVD-pNA, caspase 3 substrate, was obtained from Santa Cruz Biotechnology (Santa cruz, CA, USA). Antibodies to Syk and phospho-Syk (Y525/526) were purchased from Cell Signaling Technology (Berverly, MA, USA). Except indicated, all chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).
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!