Cyt b

Manufactured by Merck Group

Sourced in United States

Cyt B is a laboratory equipment product manufactured by Merck Group. It is designed for the measurement and analysis of cytochrome B, a fundamental component of cellular respiration. The core function of Cyt B is to facilitate the detection and quantification of cytochrome B in biological samples.

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8 protocols using cyt b

Hippocampal Glucose Uptake Assay

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As previously described, hippocampal cultures were treated with either Li₂CO₃ (hereinafter lithium, 1–50 mM_, (#255823, Sigma-Aldrich, St. Louis, MO, USA), cytochalasin B (Cyt B, 20 μM, #14930-96-2, Sigma-Aldrich), 2-deoxy-D-glucose (2-DG, 7 mM, #154-17-6, Sigma-Aldrich) or cytochalasin E (Cyt E, 20 μM, #C2149, Sigma-Aldrich) for 15 min and washed with incubation buffer (15 mM HEPES, 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, and 0.8 mM MgCl₂) [92 (link)]. Cultures were then incubated for 0–180 s with 1–1.2 μCi ¹⁴C-glucose (D-[1-¹⁴C] glucose, #NEC043X, Perkin-Elmer, Waltham, MA, USA) at a final specific activity of 1–3 disintegrations/min/pmol (~1 mCi/mmol). Glucose uptake was then arrested with detention buffer (add 0.2 mM HgCl₂ to incubation buffer (pH 7.4)) and cultures were lysed in 1 mL of lysis buffer (10 mM Tris-HCL and 0.2% SDS, pH 8.0). In total, 3 mL of scintillating solution (#LS-270, National Diagnostics, Atlanta, GA, USA) were added to the cell lysates and radioactivity was measured using a liquid scintillation counter (TriCarb 2900TR analyzer).

Gherardelli C., Cisternas P, & Inestrosa N.C. (2022). Lithium Enhances Hippocampal Glucose Metabolism in an In Vitro Mice Model of Alzheimer’s Disease. International Journal of Molecular Sciences, 23(15), 8733.

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Micronucleus Assay on Splenocytes

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Splenocytes isolated as described above were used to set up cell cultures with 2 × 10⁶ cells/mL in RPMI 1640 medium with Hepes and Glutamine (Gibco, Paisley, UK) supplemented with 1% streptomycin-penicillin, 15% inactivated calf serum and Concanavalin A (Sigma Aldrich-Merck, Milan, Italy) at a final concentration of 5 μg/mL. CytB (Sigma) was added 24 h after culture onset at a final concentration of 5 μg/mL. Binucleated cells were harvested after further 10 h of culture using a cytocentrifuge (Shandon, Waltham, MA, USA). After fixation and Giemsa staining, slides were coded and blindly scored using a brightfield microscope. The frequency of micronucleated binucleated (MnBn) splenocytes was evaluated in 1000 binucleated cells/animal. To evaluate the rate of in vitro proliferation of stimulated spleen lymphocytes the NDI (binucleated + 2 × multinucleated cells/total analyzed cells) was determined. Mean frequencies of micronucleated splenocyte in different experimental groups were compared by two-tailed Student’s t test. The limit for statistical significance was set at p = 0.05.

Russo M.T., De Luca G., Palma N., Leopardi P., Degan P., Cinelli S., Pepe G., Mosesso P., Di Carlo E., Sorrentino C., Musiani P., Crebelli R., Bignami M, & Dogliotti E. (2021). Oxidative Stress, Mutations and Chromosomal Aberrations Induced by In Vitro and In Vivo Exposure to Furan. International Journal of Molecular Sciences, 22(18), 9687.

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Micronucleus Assay Protocol for Mammalian Cells

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CBMN cytome assay was carried out as recommended by the guidelines for the in vitro mammalian cell micronucleus test [25 (link)]. Cells were exposed to cytochalasin B (Cyt B) (Sigma-Aldrich; 2.5 μg·mL⁻¹). Approximately, 150 μL of cell suspension was transferred to cytocentrifuge funnels and centrifuged for 5 min at 700 rpm to produce 1 spot per slide. Slides were removed, fixed, and stained with Instant Prov (Newprov, Pinhais, Brazil). After staining, slides were air dried and examined at 1000x magnification using a light microscope. Micronuclei (MN), nuclear buds (NBUDs), and nucleoplasmic bridges (NPBs) were counted in 1000 binucleated cells (BN) per well (4000 cells/concentration) and were scored according to Jerković et al. [30 (link)]. Cytostatic events were determined by scoring 1000 cells, including mononucleated, binucleated, and multinucleated cells, to determine cell proliferation rates as measured by the nuclear division index (NDI). Cytotoxic events were determined by the frequency of necrotic and apoptotic cells [31 (link)].

dos Santos M.S., da Silva J., Menezes A.P., de Barros F.M., Lemes M.L., Rossatto R.R., Feistel C., de Almeida I.D., Grivicich I., Prado L., Picada J.N, & de Barros Falcão Ferraz A. (2018). Biotoxicological Analyses of Trimeroside from Baccharis trimera Using a Battery of In Vitro Test Systems. Oxidative Medicine and Cellular Longevity, 2018, 7804135.

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Genotoxicity Evaluation of MCF7 and HDFa Cell Lines

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Ethanol and Polyethylene Glycol (PEG 10000N), Dulbecco's Modified Eagle's medium (DMEM), trypan blue solution, Dulbecco’s Phosphate Buffered Saline (DPBS), agarose with normal melting point and low melting point, dimethyl sulphoxide, ethidium bromide, Triton X-100, phosphate-buffered saline tablets, potassium chloride (KCl), Giemsa, ethylenediaminetetraacetic acid disodium salt dihydrate (Na2-EDTA) and cytochalasin B (Cyt-B), the positive control for the genotoxicity assays, ethyl methanesulphonate (EMS) (CAS no. 62-50-0, lot 1338043) were obtained from Sigma-Aldrich (Steinheim, Germany). Trypsin Buffer, Tyrosine Inhibitor Buffer, RNase Buffer, Propidium Iodide Stain Solutions were purchased from Becton Dickinson (BD). Sodium chloride and sodium hydroxide were purchased from Merck Chemicals (Darmstadt, Germany), whereas Chromosome medium B was purchased from Biochrom AG (Berlin, Germany). Frosted microscope slides were obtained from Menzel GmbH (Braunschweig, Germany. Human breast adenocarcinoma cell line (MCF7) and the human primary dermal fibroblast cell cultures (HDFa) were obtained from ATCC with number HTB-22 and PCS-201-12, respectively. Slides were visualized for Comet Assay by fluorescence microscopy using an Olympus BX51 System equipped with a video camera CCD-4230.

Şimşek S., Şüküroğlu A.A., Yetkin D., Özbek B., Battal D, & Genç R. (2020). DNA-damage and cell cycle arrest initiated anti-cancer potency of super tiny carbon dots on MCF7 cell line. Scientific Reports, 10, 13880.

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Characterization of TiO2 Nanoparticles Cytotoxicity

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Titanium dioxide (TiO₂ NPs) (CAS No. 13463-67-7) was purchased from Degussa-Evonik (Bitterfeld, Germany). Mitomycin C (MMC) (CAS No. 50-07-7), benzo[a]pyrene (BaP) (CAS No. 50-32-8), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (CAS No. 298-93-1), propidium iodide (PI) (CAS No. 25535-16-4) and Triton X-100 (CAS No. 9002-93-1) were purchased from Sigma-Aldrich Co. (Sintra, Portugal). Ribonuclease A from bovine pancreas (RNase A) (CAS No. 9001-99-4) was purchased Sigma-Aldrich (St. Louis, MO, USA). MMC, Act-D and BaP were dissolved in sterile distilled water, RNase A was dissolved in ultrapure water, and Cyt-B was dissolved in dimethyl sulfoxide (DMSO) (CAS No. 67-68-5), bought from Sigma-Aldrich Co. (Sintra, Portugal).

Brandão F., Fernández-Bertólez N., Rosário F., Bessa M.J., Fraga S., Pásaro E., Teixeira J.P., Laffon B., Valdiglesias V, & Costa C. (2020). Genotoxicity of TiO2 Nanoparticles in Four Different Human Cell Lines (A549, HEPG2, A172 and SH-SY5Y). Nanomaterials, 10(3), 412.

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Mitochondrial Dysfunction Assay Compounds

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Rotenone (ROT), Metformin (Met), 2-Thenoyltrifluoroacetone (TTFA), Antimycin A (AMA), Myxothiazol (MYXO), puromycin, uridine, diphenyleneiodonium chloride, ethidium bromide (EtBr), and Potassium cyanide (KCN) were purchased from Sigma-Aldrich (St. Louis, MO). The rabbit polyclonal antibody against cytochrome b (CYTB) and mouse monoclonal antibody against NDUFS1 were purchased from Sigma-Aldrich (St. Louis, MO). The rabbit monoclonal antibody against cleaved caspase-3 was purchased from Cell Signaling Technology.

Yang Q., Wang L., Liu J., Cao W., Pan Q, & Li M. (2021). Targeting the complex I and III of mitochondrial electron transport chain as a potentially viable option in liver cancer management. Cell Death Discovery, 7, 293.

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Wnt3a and Glycolysis Modulation in Neurons

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The neurons and slices were treated with recombinant Wnt3a (rWnt3a, 300 ng/mL, 0–24 h, cat: 1324-WN/CF, R&D Systems, USA), Aβ (5 μM), dickkopf-1 (Dkk1, 300 ng/mL, 0–24 h, antagonist of Wnt3a, as internal control, cat: 5897-DK/CF R&D System, USA), cytochalasin B (Cyt B, 1 μM, 3 h, inhibitor of GLUT transporters, this concentration is the IC₅₀ value, in the uptake we used 20 μM, Sigma-Aldrich USA), AZD5356 (20 nM, 12 h, an inhibitor of all Akt isoforms, cat: 5773, TOCRIS, UK), andrographolide (ANDRO, 50 μM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), carbonate of lithium (Li, 10 mM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), oligomycin (2 mM, inhibitor of ATP synthase, cat: 861944, Sigma-Aldrich USA) and 2-deoxy-D-glucose (2-DG, 7 mM, as a competitive inhibitor of hexokinase, Sigma-Aldrich USA). Neurons were carefully examined under the microscope to ensure that only plates showing uniform neuronal growth were used. After different treatments, cells were used in different metabolic assays.

Cisternas P., Zolezzi J.M., Martinez M., Torres V.I., Wong G.W, & Inestrosa N.C. (2018). Wnt-induced activation of glucose metabolism mediates the in vivo neuroprotective roles of Wnt signaling in Alzheimer disease. Journal of neurochemistry, 149(1), 54-72.

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Lymphocyte Proliferation and Micronuclei Assay

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Fresh blood was collected from 10 healthy, no smoking, no alcoholic, male donors aged between 25-35 years by venepuncture in heparinized falcons. 0.5 mL of whole blood was added to 4.5 mL of Roswell Park Memorial Institute (RPMI) culture medium 1640 supplemented with 10% fetal bovine serum containing L-glutamine, antibiotics, and phytohemagglutinin (PHA), and different doses of duloxetine (1, 10, 25, 50, 100 and 200 μL). The binucleated lymphocytes were harvested 28h after adding Cyt-B (Sigma, Missouri, USA); they were treated by hypotonic KCl (0.075M) to red blood cell (RBC) lysis. Then fixative solution (methanol: acetic acid =6:1) was added to the cells prior to slide preparation and staining. For slide preparation, 2-3 drops of cell suspension were thrown on a clean slide. The slides were stained with Giemsa solution (4%) for 7-10 mins. They were observed at 40× and 100× magnifications using a light microscope to estimate mitotic index (the cells with two or more nuclei per 1000 observed cells) and micronuclei frequency (the number of micronuclei in 1000 binucleated cells) are lymphocytes that were once divided by mitosis. The experiment was performed two times. Mitotic Index has a direct relation with cells' proliferative activity.

Shokrzadeh M., Mohammadpour A., Modanloo M., Hassani M., Barghi N.G, & Niroomand P. (2019). CYTOTOXIC EFFECTS OF ARIPIPRAZOLE ON MKN45 AND NIH3T3 CELL LINES AND GENOTOXIC EFFECTS ON HUMAN PERIPHERAL BLOOD LYMPHOCYTES. Arquivos de gastroenterologia, 56(2).

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