African green monkey kidney epithelial cell line (Vero cells) was obtained from American Type Culture Collection (ATCC) via VACSERA (Cairo, Egypt). Vero cells were cultured in DMEM medium and used as adenovirus type 7 (ADV7) and herpes simplex virus type 1 (HSV-1) host cells. While PBMCs (hepatitis C virus (HCV) host cells) were cultured in RPMI medium. Silk protein cytotoxicity was tested on both Vero cells and PBMCs using MTT assay [32 (link),33 (link)]. Briefly, both Vero cells and PBMCs were seeded in 96-well cell culture plates at densities of 104 and 105 cells/well and incubated at 37 °C in 5% CO2 for 24 h. After incubation, serial dilutions of the tested silk protein were incubated with Vero and PBMCs for 72 h. Serial dilutions of the HSV-1 and ADV7 standard drugs (ribavirin and acyclovir, respectively) were incubated with Vero cells for 72 h. At the end of incubation, 20 μL of 5 mg/mL MTT (Sigma, St. Louis, MO, USA) was added to each well and the plates were incubated at 37 °C for 3 h. After discarding MTT solution, 100 µL DMSO (dimethyl sulfoxide) was added and the dye intensity was quantified using the automated ELISA microplate reader adjusted to 570 nm to quantify the cell viability [26 (link),34 (link)]. The effective safe concentration (EC100) doses (at 100% cell viability) of the tested protein were estimated by the GraphPad Prism 9 InStat software.
Vero cells
Manufactured by Merck Group
Sourced in United States, Germany
Vero cells are a lineage of cells derived from the kidney of the African green monkey (Chlorocebus sabaeus). They are widely used in biomedical research and the production of various biologicals, including vaccines. Vero cells are known for their ability to support the growth of a variety of viruses and are commonly employed in viral propagation, assay development, and the production of viral vaccines.
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Lab products found in correlation
Vero cell, by American Type Culture Collection (7 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Hela cell, by American Type Culture Collection (2 mentions)
Jeg 3 placental, by American Type Culture Collection (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Merck Group (2 mentions)
Facscanto cytometer, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Ethylene glycol dimethacrylate egdma, by Merck Group (1 mentions)
Fourier transform infrared spectrometer, by PerkinElmer (1 mentions)
Ls 55 luminescence spectrometer, by PerkinElmer (1 mentions)
Cd no3 2 4h2o, by Merck Group (1 mentions)
Cycleavepcr mycoplasma detection kit, by Takara Bio (1 mentions)
Jcv 61v 2235 strain, by American Type Culture Collection (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Wa 1 strain, by BEI Resources (1 mentions)
Ccl 81, by American Type Culture Collection (1 mentions)
28 protocols using vero cells
1
Silk Protein Cytotoxicity Evaluation
2
Cell Death Assays by Flow Cytometry
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Apoptosis and necrosis were evaluated by staining with annexin-FITC/propidium iodide (BD Biosciences) by flow cytometry. Cells were labelled with annexin-V FITC and propidium iodide for 15 min at room temperature and analysed by flow cytometry using a BD FACSCanto cytometer and BD FACSDiva software (BD Biosciences). MTT reduction assay was performed on Vero cells following the manufacturer’s instructions (Merck).
3
Synthesis and Characterization of Polymer Nanocomposites
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Vinyl benzene and acryl amide (Merck, Germany) and ethylene glycol dimethacrylate (EGDMA) (Sigma-Aldrich, USA) were purified by distillation under reduced pressure. 2,2-(2-Methyl propionitrile) was obtained from (Arcos Organic, Geel, Belgium) and used as an initiator. Cd(NO3)2·4H2O were from (Merck, Germany). Other chemicals were of analytical grade and were purchased from (Merck, Germany).
Vero cells were received from Blood Transfusion Research Centre, Tehran, Iran.
Fourier transform infrared (FT-IR) spectroscopic measurements were performed on a PerkinElmer Fourier transform infrared spectrometer. Thermogravimetric analysis was done by TGA Model Q50 V6.3 Build189. Scanning electron microscopy (SEM) images were obtained using a Model S4160 electron micro-scope (Hitachi). The fluorescence measurements were carried out on a PerkinElmer LS55 luminescence spectrometer. UV-Vis spectra were recorded in a UV-Vis Lambda 27 spectrophotometer (PerkinElmer).
Vero cells were received from Blood Transfusion Research Centre, Tehran, Iran.
Fourier transform infrared (FT-IR) spectroscopic measurements were performed on a PerkinElmer Fourier transform infrared spectrometer. Thermogravimetric analysis was done by TGA Model Q50 V6.3 Build189. Scanning electron microscopy (SEM) images were obtained using a Model S4160 electron micro-scope (Hitachi). The fluorescence measurements were carried out on a PerkinElmer LS55 luminescence spectrometer. UV-Vis spectra were recorded in a UV-Vis Lambda 27 spectrophotometer (PerkinElmer).
4
Vero and Neuro-2a Cell Lines for Jamestown Canyon Virus
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Vero cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA; #CCL-81). Neuro-2a (N2A) cells were obtained from the JCRB cell bank (IFO50091), originating from the ATCC (#CCL-131). Vero cells were grown in Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Biowest, Nuaille, France), non-essential amino acids (Sigma-Aldrich), and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin; Thermo Fisher Scientific, Waltham, MA, USA). N2A cells were grown in DMEM supplemented with 10% heat-inactivated FBS and antibiotics. Jamestown canyon viruses (JCV; 61V-2235 strain) were purchased from the ATCC (VR-712). Mycoplasma contamination in the virus solution and cell cultures was checked using the CycleavePCR Mycoplasma Detection Kit (TaKaRa Bio Inc., Otsu, Japan) and a LightCycler 96 (Roche Life Science, Penzberg, Germany). No mycoplasma contamination was found in the virus solution and the cell culture.
5
SARS-CoV-2 Infection of iPSC-Derived Cardiac Cells
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The WA-1 strain (BEI resources) of SARS-CoV-2 was used for all experiments. All live virus experiments were performed in a Biosafety Level 3 lab. SARS-CoV-2 stocks were passaged in Vero cells (ATCC) and titer was determined via plaque assay on Vero cells as previously described7 . Briefly, virus was diluted 1:102-1:106 and incubated for 1 hour on Vero cells before an overlay of Avicel and complete DMEM (Sigma Aldrich, SLM-241) was added. After incubation at 37°C for 72 hours, the overlay was removed and cells were fixed with 10% formalin, stained with crystal violet, and counted for plaque formation. SARS-CoV-2 infections of iPSc and iPS-derived cardiac cells were done at a multiplicity of infection of 0.006 for 48 hours unless otherwise specified. For heat inactivation, SARS-CoV-2 stocks were incubated at 85°C for 5 min. Plaque assay for supernatant from infected cells was performed as above.
6
VERO Cell Propagation and Virus Production
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VERO cells used for cell propagation experiments were originally obtained from American Type Culture Collection (ATCC; CCL‐81; Manassas, VA). VERO cells were routinely cultured in flasks (Corning, Corning, NY) at 37°C/5% CO2 in humidified incubators and passaged every 3–5 days. Dengue and Zika virus (DENV and ZIKV) production evaluations were performed using VERO cells originally obtained from Sigma‐Aldrich (St Louis, MO) and maintained at the Centers for Disease Control and Prevention (CDC). A VERO‐VP30 cell line that stably expresses the VP30 gene of Zaire ebolavirus was established as previously described (Halfmann et al., 2008 ) for amplification of a biologically contained EbolaΔVP30 virus, and a working cell bank of this cell line was then established by Waisman Biomanufacturing (Madison, WI).
7
Cultivation of Vero Cells for HSV-1 Studies
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Vero cells (Sigma-Aldrich, Saint Louis, USA) were grown in DMEM supplemented with 10% FBS and 1X PenStrep antibiotic (Gibco, Life Technologies, Monza, Italy). HSV-1 strain F was a kind gift of B. Roizman (University of Chicago, Illinois, USA). Recombinant HSV-1 expressing VP16-GFP (HSV-1 [V41]) was kindly provided by Peter O’ Hare (Imperial College, London, UK).
8
Virus Cultivation and Growth Kinetics
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Two cell lines have been used for virus cultivation and growth kinetics. Ap61 cells (Aedes pseudocutellaris) were grown in L15 (Leibovitz’s 15) medium (10% heat-inactivated fetal bovine serum [FBS], 1% penicillin-streptomycin, 0.05% amphotericin B [Fungizone] (GIBCO by life technologies; USA) and 10% tryptose phosphate (Becton, Dickinson and Company Sparks, USA) and incubated at 28°C without CO2. Vero cells (African green monkey kidney epithelial cells; Cercopithecus aethiops) (obtained from Sigma Aldrich, France) were grown using the same medium without tryptose phosphate and CO2. Furthermore, PS (Porcine Stable kidney cell line, American type Culture Collection, Manassas, USA) cells were grown in same conditions than Vero cells and have been used for plaque assay.
9
Cell Culture Protocols for Placental and Epithelial Lines
10
Attenuated CHIKV strain for Maxizyme testing
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African monkey kidney (Vero) cells (ATCC, USA) were maintained on Dulbecco's modified eagle medium (DMEM; Sigma Aldrich, USA) supplemented with 10% fetal bovine serum (FBS, Atlanta Biological, Flowery Branch, GA, USA) and non-essential amino acids [(1x), Gibco, USA)]. The CHIKV 181/25 strain is an attenuated vaccine strain (a gift from Dr. Scott Weaver, UTMB, Galveston) that was used for testing the effectiveness of our maxizymes in vitro and in vivo. We chose this strain for safety reasons since we do not have appropriate containment facilities for handling a virulent strain. Additionally, while the 181/25 strain is attenuated for human virulence, it does not exhibit significant reduction in mosquito infection.
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