The gene targeted fibroblasts were inserted into enucleated oocytes by SCNT. F1 fibroblasts were isolated from 43d fetus of one pregnancy and conducted secondary SCNT using F1 fibroblasts as donors according to the protocol descrived above [17 (link)]. Consisely, oocytes enucleation was performed under UV light, and a single fibroblast cell was injected into the perivitelline space of an enucleated oocyte in M2 (Sigma, USA) medium supplemented with 10% FCS (HyClone, Beijing, China), 2 μg/mL Hoechst 33342 (Sigma, USA) and 7.5 μg/mL cytochalasin B (Sigma, USA). Fusion of cell-oocyte couplets was accomplished by providing two direct current pulses (1.5 kV/cm for 40 μs) using an electrical fusion solution containing 0.3 M mannitol, 0.5 mM HEPES, 0.05 mM CaCl2, and 0.1 mM MgSO4 (Sigma, USA). After that, reconstructed oocytes were cultured in M16 medium containing 7.5 μg/mL cytochalasin B and 5 mM ionomycin (Sigma, USA) for 5 min, and then washed with fresh M16 medium and cultured for another 5 h in M16 medium containing 7.5 μg/mL cytochalasin B and 2 mM N-6 dimethylaminopurine (Sigma, USA). Finally, obtained reconstructed embryos were maintained in M16 medium for around 10 h untill they were transplanted to a naturally cycling surrogate recipients on the second day of estrus. Recipients were subjected to a transvaginal ultrasonographic evaluation at 30 d and 60 d of gestation.
Cytochalasin b
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Japan, France, Italy, Sao Tome and Principe, Brazil, Israel, China, Canada, Spain
Cytochalasin B is a fungal metabolite that inhibits actin filament polymerization. It disrupts the cytoskeleton by binding to the barbed ends of actin filaments, preventing further addition of actin monomers. This results in the disassembly of actin-based structures within the cell.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (26 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (24 mentions)
Nocodazole, by Merck Group (23 mentions)
Giemsa, by Merck Group (18 mentions)
Potassium chloride (kcl), by Merck Group (13 mentions)
Mitomycin c, by Merck Group (11 mentions)
Acridine orange, by Merck Group (11 mentions)
Penicillin, by Thermo Fisher Scientific (11 mentions)
Colchicine, by Merck Group (10 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (10 mentions)
Phytohemagglutinin (pha), by Merck Group (10 mentions)
Methanol, by Merck Group (9 mentions)
Insulin, by Merck Group (9 mentions)
Streptomycin, by Thermo Fisher Scientific (9 mentions)
Rpmi 1640, by Thermo Fisher Scientific (8 mentions)
Hyaluronidase, by Merck Group (8 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (8 mentions)
Cytochalasin d, by Merck Group (8 mentions)
Fetal calf serum (fcs), by Merck Group (8 mentions)
Phytohemagglutinin (pha), by Thermo Fisher Scientific (8 mentions)
479 protocols using cytochalasin b
1
Cloned Fetal Fibroblasts by SCNT
2
Isolation of Cell-Derived Extracellular Vesicles
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Vesicles were isolated from MSCs (MSC CIMVs) and SNB-19 cells (SNB-19 CIMVs) using cytochalasin B (cytochalasin B from Drechslera dematioidea, #C6762-5MG, Sigma-Aldrich, St. Louis, MO, USA) as previously described [17 (link)]. When the cell culture reached a monolayer density of 90%, the medium was removed, the culture was washed twice with PBS and the cells were detached with a 0.25% trypsin-EDTA solution (PanEco, Moscow, Russia). Then the cells were washed with PBS and incubated in modified serum-free DMEM containing 10 µg/mL cytochalasin B (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C in a humidified atmosphere with 5% CO2. After incubation, the cells were vortexed for 60 s. Next, a series of subsequent centrifugations were carried out. Firstly, the cells were centrifuged at 700 rpm for 10 min, then the supernatant was collected and centrifuged at 1400 rpm for 10 min, after which the supernatant was passed through a filter (1 µm) and centrifuged at 12,000 rpm for 15 min. The precipitate containing CIMVs was washed with PBS. After centrifugation, the supernatant was removed, and the resulting CIMV pellet was dissolved in the culture medium, PBS, or another buffer, depending on the purpose of further experiments.
3
Enucleation and ICSI in Mouse Oocytes
4
Parthenogenesis of Mouse Oocytes Treated with SAL
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Mice were injected with 10 IU PMSG, followed by 10 IU human chorionic gonadotrophin (hCG, Ningbo Sansheng Pharmaceutical, Ningbo, China) 48 h later to collect MII oocytes. At 13–14 h post-hCG injection, MII oocytes were collected from the oviduct ampulla and collected in M2 medium, the cumulus cells were removed using 0.1% (w/v) hyaluronidase. Meanwhile, Different concentrations of SAL (0, 10, 20, 40 μM) were added into the medium and treated at different times (6, 12, 18, 24 h). For parthenogenesis activation, denuded oocytes were transferred first into (Ca2+)-free human tubal fluid (HTF) medium contained with 10 mmol/L strontium chloride and 5 μg/mL cytochalasin B (Merck, Darmstadt, Germany), cultured at 37 °C with 5% CO2 for 2.5 h. Then oocytes were transferred into HTF with 5 μg/mL cytochalasin B, incubated at 37 °C with 5% CO2 for 3.5 h. Activated oocytes were then cultured in KSOM plus (+) amino acids (KSOM/AA) medium (EmbryoMax® KSOM + AA with D-glucose and phenol red, EMD Millipore, Billerica, MA, USA) at 37 °C with 5% CO2 for early embryo development. Different stages of embryonic development (2-cell, 4-cell, morula and blastocyst) were recorded at 24, 48, 96, and 120 h, respectively.
5
Oocyte Activation and Embryo Development
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After in vitro maturation, the denuded oocytes were placed in a solution containing 280 mM mannitol, 0.05 mM CaCl2, 0.1 mM
MgSO4, and 0.01% BSA, and then activated by a single DC pulse of 150 V/mm for 100 µsec. The oocytes were cultured in mNCSU37 containing 0.4% BSA
and 2.2 µg/ml cytochalasin B (Sigma-Aldrich) for 3 h in order to inhibit the formation of the second polar body, then incubated for another 48 h in cytochalasin
B-free medium. Cleavage rates were calculated from the percentages of zygotes that had reached the 2-cell or a later stage at 48 h after activation.
MgSO4, and 0.01% BSA, and then activated by a single DC pulse of 150 V/mm for 100 µsec. The oocytes were cultured in mNCSU37 containing 0.4% BSA
and 2.2 µg/ml cytochalasin B (Sigma-Aldrich) for 3 h in order to inhibit the formation of the second polar body, then incubated for another 48 h in cytochalasin
B-free medium. Cleavage rates were calculated from the percentages of zygotes that had reached the 2-cell or a later stage at 48 h after activation.
6
Neurochemical Assessment of Biochemical Interactions
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Opipramol (4-[3-(5H-dibenz[b,f]-azepine-5-yl)-propyl]-1-piperazine-ethanol dihydrochloride), haloperidol, risperidone, phenelzine, pargyline, harmine, tyramine, benzylamine, semicarbazide, cytochalasin B, fatty acid-free bovine serum albumin, hydrogen peroxide, horseradish peroxidase, isoprenaline, cytochalasin B, and routinely used chemicals were obtained from Sigma-Aldrich-Merck (St. Quentin Fallavier, France), unless otherwise specified. Amplex Red was provided by InterChim (Montluçon, France). [3H]-2-Deoxyglucose, [14C]-benzylamine, and scintillation cocktail were purchased from PerkinElmer (Boston, MA, USA). Different [14C]-tyramine batches were either from PerkinElmer or Sigma. Olanzapine and ziprazidone were generous gifts from Prof. Renaud de Beaurepaire (Hosp. Paul-Guiraud, Villejuif, France).
7
Micronuclei Quantification in Binucleated Cells
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Micronuclei can be observed in binucleated cells that complete nuclear division and are blocked from cytokinesis using cytochalasin-B. Treated cells were washed, incubated with 4.5 μg/mL cytochalasin-B (Sigma) for 24 h, and harvested. Cells were then washed with PBS, treated with 0.05% KCl for 3 min at room temperature, and fixed in methanol/acetic acid (5:1, v/v) for 15 min. Cells were stained with DAPI in PBS and mounted for immunofluorescence observation. The frequency of micronuclei was assessed in 2,000 binucleated cells/treatment using an inverted microscope.
8
Foreskin Epithelial Cell Culture
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Primary cultures of foreskin epithelial cells collected from six human subjects were prepared by a two-stage enzymatic digestion as described [16 (link)] and cells were maintained in keratinocyte serum free medium (Invitrogen, Carlsbad, CA) to avoid contamination of epithelial cells with fibroblasts. Young cells started from passage 1 (cells were adapted to culture within one week after removal from liquid nitrogen). Cells were aged in vitro up to passage 8 (over 50 population doublings). The treatment of cells with cytochalasin B (Sigma Aldrich, St. Louis, MO) was done with 5 μg/mL solution of cytochalasin B in the growth medium for 12 hours.
All human tissue was obtained from the Cooperative Human Tissue Network (CHTN,http://www.chtn.nci.nih.gov ). Written informed consent was obtained when collecting tissues from patients according to their published guidelines (http://chtn.nci.nih.gov/phspolicies.html ). The purchased tissues were exempt from further IRB approval by Clarkson University since no personal information of the commercial samples was recorded and the samples were collected by the CHTN for other medical purposes according to the NIH published guidelines.
All human tissue was obtained from the Cooperative Human Tissue Network (CHTN,
9
Transgenic Goat Production via SCNT
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Somatic cell nuclear transfer (SCNT) was conducted after identifying transgene-positive clones. Enucleated oocytes served as recipients for transgenic cell nuclei, with a super electro cell fusion generator (EGFE21; nepa Gene) being used for the SCnT procedure. next, 5 µmol/l ionomycin (I0634; Sigma) and 7.5 µg/ml cytochalasin B (C6762; Sigma) in M16 medium (M7292; Sigma) was used to activate these reconstructed embryos for 5 min, and then cells were treated with M16 containing 2 mmol/l 6-dimethylaminopurine (D2629; Sigma) and 7.5 µg/ml cytochalasin B for 5 h. After activation, the embryos were implanted into recipient goats, and after a 1-month period these animals were assessed via ultrasound to confirm pregnancy. Approximately 150 days later, kids were delivered naturally. For all kids, a small portion of the ear was taken as a biopsy sample from which DNA was isolated and used to assess transgene incorporation by PCR and southern blotting. DL2000 DNA marker (3427A; Takara) was purchased from Takara Biotech (Dalian) Co., Ltd.
10
Chromosome Aberration Analysis Protocol
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For chromosome aberration analysis, colchicine (Sigma-Aldrich Co, St. Louis MO, USA) (final concentration of 0.1 μg/ml) was added to cell cultures one hour before harvesting with the conventional method. Twenty five metaphases per experimental condition were scored by recording the number of chromatid breaks, chromosome breaks and radial figures. A cytokinesis block assay, using 3 μg/ml of cytochalasin B (Sigma-Aldrich Co, St. Louis MO, USA), was implemented to obtain binucleated and tetranucleated cells: after exposing the cells to MMC for 24 h, they were washed, reincubated with fresh cytochalasin B for another 24 h and harvested using a 7:1 methanol:acetic acid fixative. Five hundred cells were scored to quantify the number of micronuclei, mononucleated, binucleated and tetranucleated cells in every experimental condition [67 (link)].
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