Dichlorodihydrofluorescein diacetate h2dcfda

Manufactured by Thermo Fisher Scientific

Sourced in United States

Dichlorodihydrofluorescein diacetate (H2DCFDA) is a cell-permeant dye that can be used to detect reactive oxygen species (ROS) within cells. It is a non-fluorescent compound that becomes highly fluorescent upon oxidation, which can be measured using a fluorescence microscope or plate reader.

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7 protocols using dichlorodihydrofluorescein diacetate h2dcfda

Apoptosis Induction and Mitochondrial ROS

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Without specific indication, the reagents obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA) were used in this study, and cell culture supplements were the products of GIBCO/Life Technologies Inc. (Carlsbad, CA, USA). ABT-263 was obtained from Apexbio Technology LLC (Houston, TX, USA), and A-1210477 was the product of MedChem Express (Monmouth Junction, NJ, USA). MitoSOX Red, tetramethylrhodamine (TMRM), annexin V-FITC/propidium iodide (PI) apoptosis detection kit, and dichlorodihydrofluorescein diacetate (H₂DCFDA) were obtained from Molecular Probes (Eugene, OR, USA); and Z-DEVD-FMK was from Calbiochem (San Diego, CA, USA).

Wang L.J., Liou L.R., Shi Y.J., Chiou J.T., Lee Y.C., Huang C.H., Huang P.W, & Chang L.S. (2020). Albendazole-Induced SIRT3 Upregulation Protects Human Leukemia K562 Cells from the Cytotoxicity of MCL1 Suppression. International Journal of Molecular Sciences, 21(11), 3907.

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Immunoblotting Analysis of Apoptosis Signaling

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Anti-PARP, anti-β-actin, anti-caspase-3, anti-caspase-9, VDAC, anti-phosphorylated ERK1/2, anti-phosphorylated p38, anti-phosphorylated JNK, anti-phosphorylated IκBα, anti-ERK1/2, anti-p38, anti-JNK, anti-Bcl-2, and anti-IκBα were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). α-Tubulin, anti-Bax (6A7) and His-probe were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against cytochrome c (for immunofluorescence, clone 6H2.B4; for western blot analysis, clone 7H8.2C12) were acquired from BD Pharmingen (San Diego, CA, USA), and the anti-cytochrome oxidase subunit IV (COX IV) antibody was purchased from Abcam (Cambridge, UK). Dichlorodihydrofluorescein diacetate (H₂DCFDA), MitoSOX, DAPI, and DiOC₆ were obtained from Molecular Probes (Eugene, OR, USA) and z-VAD-fmk, N-acetyl-cysteine (NAC), horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG, and specific inhibitors of ERK (U0126), p38 mitogen-activated protein kinase (MAPK; SB203580), JNK (SP600125), and nuclear factor-κB (NF-κB; BAY 11-7082) were purchased from Calbiochem (San Diego, CA, USA). Anti-F4/80 and anti-CD11b were performed as recommended by the manufacturer (eBioscience, San Diego, CA, USA).

Lee K.I., Whang J., Choi H.G., Son Y.J., Jeon H.S., Back Y.W., Park H.S., Paik S., Park J.K., Choi C.H, & Kim H.J. (2016). Mycobacterium avium MAV2054 protein induces macrophage apoptosis by targeting mitochondria and reduces intracellular bacterial growth. Scientific Reports, 6, 37804.

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Detection of Reactive Species in Gelatin

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Detection of reactive species in gelatin samples was performed similarly as described elsewhere (Dobrynin et al., 2012 ; Park et al., 2013 ). Hydrogen peroxide was detected by measuring fluorescence of 100 µm solution of Amplex UltraRed reagent in PBS (Molecular Probes, Eugene, OR, USA) at an excitation frequency of 530 nm and emission at 590 nm, with 100 U/µl of H₂O₂ (MP Biomedicals, Santa Ana, CA, USA) in 10% gelatin prepared in PBS. A 100-µl aliquot of reagent solution was added to warm liquid gelatin (1 ml), poured into six-well plates and incubated in the dark for 10 min at room temperature until solidification. Samples were then treated with DBD at fixed 2 mm distance and fluorescence was measured 5 min afterwards using an LS55 (Perkin Elmer) fluorescent spectrometer. Nitric oxide was detected using a similar procedure. For that, 100 µl diaminofluorescein-2 (DAF-2) reagent (50 µm in dimethylsulphoxide (DMSO) excitation frequency 485 nm, emission frequency 538 nm; Cayman Chemical), was added to gelatin before solidification. Dichlorodihydrofluorescein diacetate (H₂DCFDA, 50 µl in DMSO excitation frequency 495 nm, emission frequency 518 nm; Molecular Probes) was used to detect peroxynitrite anion (ONOO⁻) in gelatin. In addition, DMSO and horseradish peroxidase (100 U) were used as hydroxyl radical (OH) and H₂O₂ scavengers.

Eisenhauer P., Chernets N., Song Y., Dobrynin D., Pleshko N., Steinbeck M.J, & Freeman T.A. (2016). Chemical modification of extracellular matrix by cold atmospheric plasma-generated reactive species affects chondrogenesis and bone formation. Journal of tissue engineering and regenerative medicine, 10(9), 772-782.

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ROS Measurement in Rat MBH

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One minute after the beginning of the carotid glucose injection, or five minutes after rotenone injection, rats were decapitated, brains quickly removed, and the MBH dissected, frozen in liquid nitrogen, and then stored at −80 °C. ROS determination was performed as described previously [27] (link) using the fluorescent probe dichlorodihydro-fluorescein diacetate (H₂-DCFDA; Molecular Probes, Eugene, OR, USA) that primarily detects H₂O₂ levels. ROS measurements were performed in a fluorescent plate reader (Perkin Elmer, Courtaboeuf, France) at 535 nm, under excitation at 490 nm. Intensities of fluorescence were calculated as arbitrary units per milligram of protein.

Desmoulins L., Chrétien C., Paccoud R., Collins S., Cruciani-Guglielmacci C., Galinier A., Liénard F., Quinault A., Grall S., Allard C., Fenech C., Carneiro L., Mouillot T., Fournel A., Knauf C., Magnan C., Fioramonti X., Pénicaud L, & Leloup C. (2018). Mitochondrial Dynamin-Related Protein 1 (DRP1) translocation in response to cerebral glucose is impaired in a rat model of early alteration in hypothalamic glucose sensing. Molecular Metabolism, 20, 166-177.

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Curcumin and Cisplatin Synergistic Apoptosis

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Curcumin and cisplatin were purchased from Selleckchem (Houston, TX). Z-VAD-FMK (a pan-caspase inhibitor) and U0126 (an ERK inhibitor) were purchased from Calbiochem (San Diego, CA, USA). NAC (a ROS scavenger) was purchased from Sigma Chemical Company (St. Louis, MO, USA). Fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI) were purchased from BD Biosciences Pharmingen (San Diego, CA, USA). Dichlorodihydrofluorescein diacetate (H2DCFDA) was purchased from Molecular Probes (Waltham, MA, USA). Rabbit polyclonal antibodies against caspase-3, Bcl-2, p-MEK, MEK, p-ERK1/2, and ERK were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse polyclonal antibodies against p53 and p21 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Mouse monoclonal antibody against XIAP was purchased from BD Biosciences Pharmingen (San Diego, CA, USA).

Park B.H., Lim J.E., Jeon H.G., Il Seo S., Lee H.M., Choi H.Y., Jeon S.S, & Jeong B.C. (2016). Curcumin potentiates antitumor activity of cisplatin in bladder cancer cell lines via ROS-mediated activation of ERK1/2. Oncotarget, 7(39), 63870-63886.

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Influenza virus infection and ginseng extract

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The influenza subtype H1N1 A/PR/8/34 (A/PR8) and A/WSN/1933 viruses were described in previous studies [13 (link),14 (link)]. Influenza viruses were grown in 11-day old embryonated hen’s eggs. Egg allantonic fluids were harvested and stored at −80 °C until use. A549 cells, a human alveolar type II-like epithelial cell line, were generous gifts from Dr Jae Hyang Lim (Center for Inflammation, Immunity & Infection, Georgia State University). Mice were infected with serial dilutions of influenza virus and the 50% lethal dose (LD₅₀) was determined. Korean red ginseng extract (RGE), a concentrated form of the commercial ginseng product was kindly provided by Korea Ginseng Corporation (Daejeon, Korea). Briefly, fresh roots of the Panax ginseng that had grown for 6 years were washed, steamed at 100 °C for 2 to 3 h and dried. The dried red ginseng roots were boiled in 4 to 5 volumes of water for 3 h and the supernatants were concentrated. This preparation was designated “RGE” (approximately 36% water content). Fetal bovine serum (FBS), penicillin–streptomycin, and F12K Nutrient Mixture were purchased from GIBCO (Grand Island, NY, USA). Dichlorodihydrofluorescein diacetate (H₂DCFDA) was purchased from Molecular Probes (Carlsbad, CA, USA). All other chemicals were analytical grade.

Lee J.S., Hwang H.S., Ko E.J., Lee Y.N., Kwon Y.M., Kim M.C, & Kang S.M. (2014). Immunomodulatory Activity of Red Ginseng against Influenza A Virus Infection. Nutrients, 6(2), 517-529.

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Protease-activated receptor 2 analysis

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Unless stated otherwise, all other chemicals were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Mouse monoclonal antibody (Ab) against human protease-activated receptor 2 (PAR2) (SAM 11) and normal mouse IgG_2a were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fluorescent isothiocyanate (FITC)-labeled annexin V, PE-conjugated anti-human CD63, and PE-conjugated anti-mouse IgG₁κ were purchased from BD Pharmingen (San Diego, CA, USA). Dichlorodihydrofluorescein diacetate (H₂DCFDA) was purchased from Molecular Probes (Eugene, OR, USA).

Lee Y.A., Nam Y.H., Min A., Kim K.A., Nozaki T., Saito-Nakano Y., Mirelman D, & Shin M.H. (2014). Entamoeba histolytica-secreted cysteine proteases induce IL-8 production in human mast cells via a PAR2-independent mechanism. Parasite, 21, 1.

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