The antibodies against NOX4, P38, JNK, OPN, MCP-1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from OriGene Technologies (Rockville, MD, USA). Goat anti-rabbit and goat anti-mouse immunoglobulin G, protein extraction reagent, were purchased from CW Biotech (Beijing, China). GKT137831was purchased from Selleck Chemicals (Selleck Chemicals, Houston, TX, USA), while phorbol-12-myristate-13-acetate (PMA) and dichlorodihydrofluorescein diacetate (DCFH-DA) were acquired from Beyotime Biotechnology (Beyotime Institute of Biotechnology, Shanghai, China). Furthermore, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin–streptomycin solution were obtained from HyClone (Thermo Fisher Scientific, Waltham, MA, USA). Dimethyl sulfoxide (DMSO) was provided by MP Biomedicals, LLC (MP Biomedicals, LLC, Santa Ana, CA, USA). OA was donated by Dr. Meigeng Hu from the Institute of Medicinal Plant Development, and the purity was 95%. In cell experiments, OA was dissolved in DMSO to prepare a 20 mmol solution, which was diluted to the desired concentration using DMEM medium. In DPPH radical scavenging assay, OA was dissolved in ethanol.
Dichloro dihydro fluorescein diacetate dcfh da
Manufactured by Beyotime
Sourced in China
Dichloro-dihydro-fluorescein diacetate (DCFH-DA) is a cell-permeable fluorogenic probe used for the detection of reactive oxygen species (ROS) in biological systems. It is a non-fluorescent compound that becomes highly fluorescent upon oxidation, allowing for the measurement of intracellular ROS levels.
Automatically generated - may contain errors
Lab products found in correlation
Atp assay kit, by Beyotime (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Gkt137831, by Selleck Chemicals (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Beyotime (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Gapdh, by OriGene (1 mentions)
Dimethyl sulfoxide (dmso), by MP Biomedicals (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
Rapamycin, by MedChemExpress (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
P erk thr202 tyr204, by Cell Signaling Technology (1 mentions)
P p38 thr180 tyr182, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
P38 t p38, by Cell Signaling Technology (1 mentions)
T akt, by Cell Signaling Technology (1 mentions)
T erk, by Cell Signaling Technology (1 mentions)
Cell counting kit 8 cck 8 assay, by Beyotime (1 mentions)
Z lehd fmk, by Selleck Chemicals (1 mentions)
Imidazole ketone erastin, by Selleck Chemicals (1 mentions)
Poly adp ribose polymerase parp, by Cell Signaling Technology (1 mentions)
17 protocols using dichloro dihydro fluorescein diacetate dcfh da
1
Antioxidant and Anti-inflammatory Assays
2
ROS Detection using DCFH-DA
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Dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime, Shanghai, People’s Republic of China) was used to detect ROS production according to the manufacturers’ protocol. Briefly, 5 * 105 cells were planted in the 6-well plate in different conditional culture medium (100 μM FAC or 100 μM DFO) for 24 h. at the day of measurement, then the culture medium was removed. Next, FBS-free medium with DCFH-DA was added to the dish and then incubated for 20 min. The fluorescence intensity of cells was detected by microplate reader.
3
Synthesis and Characterization of Curcumin Acetylsalicylate
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Curcumin acetylsalicylate (CA, mass fraction > 98%) was synthesized by acyl chloride reaction in the College of Pharmacy, Hunan University of Chinese Medicine, China [19 (link)]. Rapamycin from MedChemExpress LLC (Beijing, China), paraquat-dichloride (C36H32F24N4P4), and levamisole hydrochloride were purchased from Solarbio Life Sciences (Beijing, China). Rapamycin or CA was dissolved in dimethyl sulfoxide (DMSO) with final concentrations of less than 0.1% in all experiments, which did not have any significant effect on the lifespan of worms [20 (link)]. Paraquat and levamisole hydrochloride were dissolved in distilled water. The ROS assay kit and the dichloro-dihydro-fluorescein diacetate (DCFH-DA) were purchased from Beyotime Biotechnology (Shanghai, China).
4
Exploring Antioxidant and Apoptotic Pathways
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The antagomir and agomir of miR-143 and the negative controls were purchased from RiboBio (Guangzhou, China). Doxorubicin was obtained from Sigma-Aldrich (St. Louis MO, USA). Measurement of levels of malonaldehyde (MDA), and the antioxidant, glutathione (GSH), and the activity of superoxide dismutase (SOD) and NADPH oxidase (NOX) were detected using kits and were performed at the Nanjing Jiancheng Bioengineering Institute. The levels of 3-nitrotyrosine (3-NT) and 4-hydroxynonenal (4-HNE) were quantified using enzyme-linked immunosorbent assay (ELISA) kits that were purchased from Abcam (Cambridge, MA, USA). The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) fluorescence detection kit was purchased from Merck Millipore (Burlington, MA, USA). The cell counting kit-8 (CCK-8) assay and dichloro-dihydro-fluorescein diacetate (DCFH-DA) were obtained from Beyotime Biotechnology (Shanghai, China). The following primary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA) and included antibodies to phosphorylated P38 MAP kinase (P-P38, Thr180/Tyr182), total P38 (T-P38), P-extracellular signal-regulated kinase (P-ERK, Thr202/Tyr204), T-ERK, protein kinase C epsilon (PKCɛ), P-protein kinase B (P-AKT, Ser473), T-AKT, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
5
Cyclic AMP Signaling and Autophagy
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The cyclic adenosine monophosphate in oocytes was a derivative of adenosine triphosphate (ATP), which serves as the second important messenger in many biological processes. Further, cAMP signaling participates in the process of autophagy via a novel pathway including ERK, cyclin E, and Beclin 1 [38 (link)]. Thus, the level of cAMP was detected using luciferase. Further, the ATP level was measured with the ATP Assay Kit (Beyotime, Haimen, China) according to the manufacturer’s instructions. The ROS level in the mitochondria was detected with dichloro-dihydro-fluorescein diacetate (DCFH-DA) and probes (Beyotime, Haimen, China) as described in a previous work [39 (link)]. In brief, oocytes were washed three times with PBS buffer, then suspended with 10-μM DCFH-DA, and incubated at 37°C for 30 min. Finally, they were rinsed with the PBS buffer thrice. The fluorescence intensity was measured at the wavelengths of 488 and 525 nm.
6
Ferroptosis Induction and Regulation
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Z-VAD-FMK, 3-methyladenine (3-MA), deferoxamine and RSL3 were obtained from MedChemExpress (NJ, USA); Z-LEHD-FMK and imidazole ketone erastin (IKE) were purchased from Selleck Chemicals (Houston, USA). Dichlorodihydro-fluorescein diacetate (DCFH-DA) was purchased from Beyotime Biotechnology (Shanghai, China). Antibodies against BCL-2, BAX, caspase 9, caspase 3, poly ADP-ribose polymerase (PARP) and β-actin were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against SLC7A11 and GPX4 were obtained from Abcam (Cambridge, UK). Antibody against GAPDH was acquired from Goodhere Biological Technology (Hangzhou, China).
7
Comprehensive Immunological and Biochemical Profiling
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Alhydrogel® adjuvant 2% was purchased from InvivoGen (San Diego, CA, USA). Brain Heart Infusion (BHI) Broth and BHI Agar were purchased from Qingdao Hope Bio-Technology Co., Ltd. (Qingdao, China). Crystal violet was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Dichloro-dihydro-fluorescein diacetate (DCFH-DA) was purchased from Beyotime Biotechnology (Shanghai, China). Masson’s trichrome staining kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Red blood cell lysis buffer and 4′,6-diamidino-2-phenylindole were purchased from Beyotime Biotechnology (Shanghai, China). All antibodies used in flow cytometry were purchased from BioLegend (San Diego, CA, USA). Interferon γ (IFNγ) and interleukin (IL)-4 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Cusabio Biotechnology Co., Ltd. (Wuhan, China). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), blood urea nitrogen (BUN), total cholesterol (TC), triglyceride (TG), and glucose (GLU) assay kits were purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China).
8
Antibacterial Activity Evaluation of Phytochemicals
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The standard compounds of 6-prenylnaringenin, licoisoflavone B, glabrone, licochalcone B, licochalcone D, isobavachin, corylifol A, bavachin, isobavachin, and bavachalcone were purchased from Yuanye Co., Ltd. (Shanghai, China). Dichloro-dihydro-fluorescein diacetate (DCFH-DA) and ATP Assay Kit were purchased from Beyotime (Shanghai, China). 3,3′-dipropylthiadicarbocyanine iodide [DiSC3(5)], propidium iodide (PI), and naphthylamine were purchased from Aladdin (Shanghai, China). Other reagents were all commercially available. The tested strains included S. aureus (ATCC 25923), S. epidermidis (CMCC 26069), E. coli (CICC 20658), and Shigella dysenteriae (CICC 23829). MRSA and Pseudomonas aeruginosa were two clinically isolated bacteria donated by Nanjing Medical University.
9
Photofrin-mediated Photodynamic Therapy
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Photofrin® (75mg/vail) was purchased from Union Med. Group Limited Company, Hong Kong, China. The reagents for cell culture were obtained from Life Technology. Doxycycline (Dox) and puromycin were obtained by Abcam. Fluo-3-Am and dichlorodihydrofluorescein diacetate (DCFH-DA) were from Beyotime, China. 4-HNE and ceramide ELISA kits were supplied by Shanghai Enzyme-linked Biotechnology Co., Ltd, China. Other reagents were all supplied by Sigma-Aldrich unless otherwise stated.
10
Intracellular ROS Measurement by DCFH-DA
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The dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime Institute of Biotechnology, China) was utilized to test intracellular ROS. The final concentration of DCFH-DA was adjusted to 10 mol/L via serum-free medium with 1:1,000. ccRCC cells transfected with sh-UCN and shControl plasmids were inoculated into 96-well plates (10,000 cells per well). Cells were washed with serum-free medium for three times after incubation at 37 °C for 30 min. Finally, the excitation fluorescence values were measured at 488 nm, while the emission fluorescence values were measured at 525 nm through a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).
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