Cells were plated at a density of 2.6 × 104 cells/cm2 in growth medium on a 24-well plate and grown for 3 days at 33°C. The growth medium was replaced and the cells were cultured for an additional 2 days under 37°C and treated with either 10 nM hPTH(1–34) or 10 μM forskolin for 18–22 hrs followed by an exposure to 1 mM H2O2 for 4 hrs or overnight. Cell viability was measured using PrestoBlue cell viability reagent as per manufacturer’s instruction. Intracellular levels of ROS were determined upon cell staining with 14 μM of 2′, 7′-dichlorofluorescin diacetate (DCFDA) (Sigma) for 30 min at 37°C with 5% CO2. The cells were then trypsinized and transferred into wells of a 96-well plate and fluorescence (485 nm excitation and 535 nm emission) was measured using a spectrophotometer (TriStar LB941, Berthold Technologies, Oak Ridge, TN, USA). The fluorescent intensity was normalized with cell viability.
2 7 dichlorofluorescin diacetate dcfda
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
2',7'-dichlorofluorescin diacetate (DCFDA) is a non-fluorescent cell-permeable compound that can be used to detect the presence of reactive oxygen species (ROS) within cells. Upon oxidation by ROS, DCFDA is converted into a highly fluorescent compound, 2',7'-dichlorofluorescein (DCF), which can be detected using fluorescence-based methods.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (14 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Propidium iodide, by Merck Group (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (3 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (3 mentions)
Mitosox red, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
N acetyl cysteine (nac), by Merck Group (2 mentions)
Filipin 3, by Merck Group (2 mentions)
N acetyl l cysteine nac, by Merck Group (2 mentions)
Cresyl violet acetate, by Thermo Fisher Scientific (2 mentions)
Zncl2, by Thermo Fisher Scientific (2 mentions)
3 4 5 dimethylthiazol 2 yl 5 3 carboxymethoxyphenyl 2 4 sulfophenyl 2h tetrazolium mts reagent, by Promega (2 mentions)
Monochlorobimane mcb, by Thermo Fisher Scientific (2 mentions)
Cuso4 5h2o, by Thermo Fisher Scientific (2 mentions)
92 protocols using 2 7 dichlorofluorescin diacetate dcfda
1
Oxidative Stress Response in Bone Cells
2
Intracellular ROS Generation in hSod1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The generation of intracellular reactive oxygen species (ROS) was assessed in H4 cells expressing BiFC-tagged hSod1 or untagged hSod1 (WT and mutants). Briefly, the cells were seeded in 48-well plates and washed once with DPBS. For ROS production measurement, cells were incubated with 25 μM 2′,7′-dichlorofluorescin diacetate (DCFDA, Sigma) at 37 °C for 30 min. Following the incubation period, two washing steps with DPBS were performed to remove excess probe and fluorescence intensity (excitation 485 nM; emission 535 nM) was measured using the microplate reader Infinite M2000 PRO, Tecan. After three basal measurements, cells were challenged with 5% (v/v) H2O2. The fluorescence values were recorded up to 45 min and normalized to those obtained from non-H2O2-induced cells.
3
Assessing Cellular ROS Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ROS levels inside the cells were measured with 2′,7′‐dichlorofluorescin diacetate (DCFDA; Sigma‐Aldrich) reagent after 24 h of Hg2+ exposure. Once diffused into the cell and de‐esterified by cellular esterase, DCFDA is oxidized to highly fluorescent 2′,7′‐dichlorofluorescin (DFC) by ROS (Rosenkranz et al., 1992 (link)). The exposure conditions were identical to those of the cytotoxicity and GSH assays (also with or without poly I:C and BSO). Cells were first exposed for 20 h and then labeled with 25 µM DCFDA for another 4 h before fluorescence measurement at ex/em 485/535 nm. Results are presented as ratios relative to the negative controls in the –BSO –poly I:C group.
4
GHK-Cu Modulates LPS-Induced Inflammatory Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GHK-Cu (MW 340.38; soluble in water) was provided by Bioceltran Corporation Ltd., (Chuncheon, Korea). Lipopolysaccharide (Escherichia coli O111:B4), thioclycollate and 2′,7′-dichlorofluorescin-diacetate (DCFDA) were purchased from Sigma (MO, USA). Antibodies against phospho-ERK1/2 (#4370), total ERK1/2 (#4695), phospho-p38 MAPK (#4511), total p38 MAPK (#9212), phospho-JNK1/2 (#4668), total JNK1/2 (#9252), phospho-NF-κB p65 (Ser536, #3033) and β-actin (#3700) were purchased from Cell Signaling (MA, USA). Antibodies against NF-κB p65 (sc-372) was purchased from Santa Cruz Biotechnology (CA, USA).
5
Oxidative Stress and Inflammatory Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ethylenediaminetetraacetic acid disodium salt (Na2-EDTA), sodium azide, LPS and 2,7-dichlorofluorescin diacetate (DCF-DA) were purchased from Sigma Chemical Co (St. Louis, MO, USA). The materials needed for cell culture were obtained from Gibco BRL (Gaithersburg, MD, USA), and VECTASHIELD mounting medium for fluorescence staining with DAPI was purchased from Vector Laboratories Inc (Burlingame, CA, USA). JNK, COX-2 and GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The ECL kit was purchased from Amersham BioScience (Amersham, UK) and probenecid was obtained from Sigma Chemical Co.
6
Evaluation of Antioxidant Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LA, CA, NAC, Y-27632 (Y) and 2′,7′-dichlorofluorescin diacetate (DCFDA) were purchased from Sigma-Aldrich (Milan, Italy). Tyrphostin AG1478 (AG), PD98059 (PD), SP 600125 (SP) and UC2288 were obtained from Calbiochem (DBA, Milan, Italy). All compounds were dissolved in dimethyl sulfoxide except LA, CA, NAC and Y-27632 (Y), which were solubilized in water.
7
Curcumin-Loaded PLGA Nanoparticle Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Curcumin (95% purity) was obtained from Alfa Aesar (Ward Hill, MA, USA); PLGA (Resomer RG 503 H) was supplied by Evonik (Essen, Germany); poly(vinyl alcohol) (PVA, Mowiol 4-88) was purchased from Kuraray (Hattersheim, Germany); polysorbate 80 (Tween 80), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2’,7’-dichlorofluorescin diacetate (DCFDA) and tert-butyl hydroperoxide (TBHP) were obtained from Sigma-Aldrich Chemie (Taufkirchen, Germany). Inhibitors of cellular uptake, viz. Filipin III, chlorpromazine and dynasore were also obtained from Sigma Aldrich. Ultrapure water, generated by a PURELAB flex 4 device (ELGA LabWater, High Wycombe, UK) was used for all experiments. For cell culture studies, ultrapure water was additionally autoclaved and filter-sterilised using 0.2 μm polyethersulphone membrane filters (Sarstedt, Nümbrecht, Germany) prior to use. HPLC-grade ethyl acetate (VWR, Darmstadt, Germany) was used to prepare the organic phases. All other chemicals used were of analytical grade. All buffers used in this study were prepared in the laboratory, unless stated otherwise.
8
Comparative Investigation of Anti-Cancer Agents
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ALLN (#sc-221236), pifithrin-alpha (PFT-α, #sc-45050), and rapamycin (Rapa, #sc-3504A) were purchased from Santa Cruz Biotechnology. CDDP (#232120) was obtained from MERCK. 2′,7′-dichlorofluorescin diacetate (DCFDA, # D6883), monodansylcadaverine (MDC, # D4008), CQ (#C6528), and propidium iodide (PI; #P4864) were purchased from Sigma; N-Acetyl-L-cysteine (NAC, #47866) and 3-(4, 5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT, #33611) were obtained from SRL. Geneticin (G418, # 10131-035), FITC conjugated Annexin V (#A13199), Annexin V binding buffer (#V13246), and LysoTracker Green DND-26 (# L7526) were procured from Thermo Fisher Scientific. Lipofectamine 3000 was from Invitrogen (#L3000-001). The MAPK inhibitor, U0126, and gene specific primary and secondary antibodies were obtained from Cell Signaling Technology (CST, USA). pCMV-Neo-Bam p53 wt (Addgene Plasmid #16434), pCMV-Neo-Bam p53 R273H (Addgene Plasmid #16439), and pCMV-Neo-Bam Empty Vector (Addgene Plasmid # 16440 ) were a gift from Bert Vogelstein.
9
Mitochondrial Dysfunction and Oxidative Stress Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Irinotecan hydrochloride, 2-thenoyltrifluoroacetone (TTFA), hydroxyurea (HU), 2′,7′-dichlorofluorescin diacetate (DCFDA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2-deoxy-d -glucose (2-DG), crystal violet, and antimycin A were purchased from Sigma Aldrich (Deisenhofen, Germany). Doxorubicin was purchased from Enzo Life Science (Lörrach, Germany). Chloramphenicol was purchased from Carl Roth GmbH (Karlsruhe, Germany). Rotenone, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and oligomycin were purchased from Abcam (Cambridge, UK). 3,3′-Dihexyloxacarbocyanine iodide (DiOC6(3)) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). DMSO was used as a treatment control.
10
Mitochondrial Membrane Potential and ROS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondrial membrane potential ΔΨm was measured by the sensitive and relatively mitochondrion-specific lipophilic cationic probe fluorochrome 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazoly-carbocyanine iodide (JC-1) (Molecular Probes, Eugene, OR, USA). Briefly, HKC8 cells were incubated with JC-1 (5 µmol/L) at 37 °C for 20 min and examined by flow cytometry (BD FACSCaliber Flow Cytometry, San Jose, CA, USA). Intracellular ROS was measured by 2′,7′-dichlorofluorescin diacetate (DCF-DA) (Sigma, St. Louis, MO, USA). Briefly, HKC8 cells were incubated with DCF-DA (5 µM) at 37 °C for 30 min and examined by flow cytometry (BD FACSCaliber Flow Cytometry, San Jose, CA, 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!