The heparinized blood samples were collected from four healthy males aged from 25 to 30 years old, respectively, non-smoking, non-alcoholic, not under drug therapy and with no recent history of exposure to mutagens. Human peripheral blood cultures were set up according to a slight modification of the protocol described by Evans and O’Riordan (1975[7 (link)]). The heparinized blood (0.5 ml) was cultured in 6 ml of culture medium (Chromosome Medium B, Biochrom, Berlin, Germany) with 5 mg/ml of phytohemagglutinin (Biochrom). Potassium tetraborate (K2B4O7, CAS Number 12045-78-2) was synthetized previously described by Marezio et al. (1963[15 ]) and dissolved in distilled water and it was added to the cultures just before incubation in concentrations of 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320, 640 and 1280 µg/ml. Each individual blood culture without PTB was studied as a control group. The concentrations were selected according to the works of Turkez et al. (2007[25 ]). Mitomycin C (MMC; 10-7 M, Sigma-Aldrich®) was used as the positive control in CA and MN assays. Ascorbic acid (10 µM, Sigma-Aldrich®) and hydrogen peroxide (25 µM, Sigma-Aldrich®) were also used as the positive controls in TAC and TOS analysis, respectively.
Chromosome medium b
Manufactured by Harvard Bioscience
Sourced in Germany
Chromosome Medium B is a specialized laboratory medium used for the cultivation and growth of chromosomal materials. It provides the necessary nutrients and conditions to support the maintenance and proliferation of chromosomes during scientific experiments and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Phytohemagglutinin (pha), by Harvard Bioscience (2 mentions)
Mitomycin c mmc, by Merck Group (2 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Phosphate buffered saline tablet, by Merck Group (1 mentions)
Dimethyl sulphoxide, by Merck Group (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Bx51 system, by Olympus (1 mentions)
Cyt b, by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
Trypan blue solution, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Penicillin, by Harvard Bioscience (1 mentions)
Streptomycin, by Harvard Bioscience (1 mentions)
Cytochalasin b, by Merck Group (1 mentions)
5 protocols using chromosome medium b
1
Cytogenetic Evaluation of Potassium Tetraborate
2
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.
3
Lymphocyte Culture Protocol with DMAA and cDMAA
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The CA method for lymphocyte culture was performed with slight modifications of the previous procedure [33] (link). The blood sample (0.5 mL) and the concentrations described above of DMAA and cDMAA were cultured with 6 mL of Chromosome Medium B (Biochrom, Berlin) for 72 h at 37 °C. Two hours before the end of the incubation period, colchemide solution (0.1 mL) was added to the culture. After the incubation period, cells were collected by centrifugation and treated with a hypotonic solution (0.075 M KCl). Cells were re-incubated and centrifuged. A fixation solution (methanol to acetic acid, 3:1 v/v) was added to the cell suspension, and the resulting cells were resuspended and dropped onto clean slides. To prepare slides, a few drops of the fixed cell suspension were dropped onto the cold slide and air-dried. The slides were stained with Giemsa stain in phosphate buffer (pH 6.8) and allowed to dry. The evaluation process was performed by counting the fifty-metaphase plate showing different chromosome anomaly.
4
Micronucleus Assay with Ochratoxin A and Bisphenol A
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MN assay was carried out under procedures previously described by Robbiano et al. [32] (link). Five hundred microliter of the blood sample and OTA (25 μM), BA (0.5, 5 and 50 μM), and their combinations were added to 7 mL of Chromosome Medium B (Biochrom, Leonorenstr. 2-6.D-12247, Berlin) containing 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.5 mL of phytohemagglutinin (Biochrom) and cell culture was incubated at 37 °C for 72 h. Cytochalasin B (Sigma) was added to the culture medium at 44 h of incubation. After the incubation period, the culture medium was centrifuged at 900 × g for 10 min and lymphocyte cells obtained were hypotonized by 0.075 M of cold potassium chloride for 30 min and then cells were fixed with ice-cold methanol/ acetic acid (3:1, v/v). The fixed cells were placed directly on slides using a cytospin and stained with Giemsa solution. The count of MN cells was performed under a light microscope by criteria declared by Robbiano et al. [32] (link). At least 2000 binucleated cells were counted per concentration (duplicate cultures for each concentration) for the formation of one, two, or more MN.
5
Nanoparticle Genotoxicity Evaluation in Human Blood
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This research was carried out using heparinized human blood samples from 4 healthy, nonsmoking donors (male) aged between 20 and 24 years, and they were not exposed to radiation by their occupation. Hematological and biochemical parameters of volunteers were analyzed, and no pathology was detected. Informed consent forms were signed by each donor. Cultures were set up according to a previously published protocol with a slight modification. 10 The heparinized whole blood samples (0.5 mL) were cultured in 6 mL of culture medium (Chromosome Medium B, Biochrom, United Kingdom) with phytohemagglutinin. Nanoparticles suspensions (0.5 mL) were added to blood culture tubes to achieve final concentrations of 1, 12.5, 25, 50, 100, 250, 500, 1000, and 2000 ppm. The negative and the positive control groups were distilled water and mitomycin C (MMC; at 10 À7 M, Sigma), respectively. The blood cultures were incubated in complete darkness for 72 hours at 37 C in an incubator.
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