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Vero e6

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The Vero E6 is a cell line derived from the kidney epithelial cells of an African green monkey. It is commonly used in research applications, particularly for the study of viral infections and vaccine development.

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15 protocols using vero e6

1

Cell Culture and Virus Propagation

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Cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Serum Plus II, MilliporeSigma), 10 mM HEPES, 50 U/mL penicillin-streptomycin (Gibco), and 0.05 mM β-mercaptoethanol. HCT-8, Calu-3, and VeroE6 cells were obtained from the American Type Culture Collection. 293T-ACE2 cells that overexpress ACE2 (28 (link)) were a kind gift from Dr. Jesse D. Bloom (Fred Hutchinson Cancer Research Center, Seattle, WA, USA). HCoV-OC43 (NR-52725) was obtained through BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) and propagated in HCT-8 cells using medium containing 2% FBS. HCoV-OC43 was titered in HCT-8 cells using a focus-forming assay and used for infections at an MOI of 0.01, unless otherwise indicated. SARS-CoV-2 Isolate USA-WA1/2020 (NR-52281) was obtained through BEI Resources, NIAID, NIH; propagated in VeroE6 cells; titered in VeroE6 cells using a plaque-forming assay; and used for infections at an MOI of 0.01, unless otherwise indicated. Cells were treated with small-molecule IRE1α inhibitors for 2.5 hours prior or tBHP for 2 hours prior to infection.
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2

SARS-CoV-2 Intranasal Infection in Mice

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SARS-CoV-2 virus strain USA-WA1/2020 (P4, Catalog#: NR-53873, Lot#:70039812, 9.2X105 TCID50/ml (Vero E6) Lot#:70039812) was obtained from BEI Resources. SARS-CoV-2 was gently thawed on ice and used to directly inoculate mice. Mice were anesthetized by the inhalation of vaporized isoflurane and intranasally infected with 50 μl of the virus stock to a final infection dose of 4.6 × 104 (link) TCID50.
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3

SARS-CoV-2 USA-WA1/2020 Isolation and Characterization

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African green monkey kidney epithelial cells (Vero E6, CRL-1586) were obtained from the American Type Culture Collection (ATCC; Bethesda, MD) and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% (vol/vol) fetal bovine serum (FBS; VWR) and 100 units/ml penicillin-streptomycin (Corning).
The SARS-CoV-2 USA-WA1/2020 natural isolate was obtained from BEI Resources (NR-52281) and amplified on Vero E6 cells. This strain was selected because it was isolated from an oropharyngeal swab from a patient with respiratory illness in January 2020 in Washington, DC. The SARS-CoV-2 USA-WA1/2020 sequence was available from GenBank (accession no. MN985325).
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4

SARS-CoV-2 Amplification in Vero E6 Cells

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Vero E6 (CRL-1586; American Type Culture Collection) were cultured Dulbecco’s Modified Eagle’s Medium (DMEM, Corning) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologics) and 1% nonessential amino acids (NEAA, Corning). SARS-CoV-2, isolate USA-WA1/2020 19 (BEI resources #NR52281), a gift from Dr. Mark Mulligan at the NYU Langone Vaccine Center was amplified once in Vero E6cells. All experiments with SARS-CoV-2 were conducted in the NYU Grossman School of Medicine ABSL3 facility in accordance with its Biosafety Manual and Standard Operating Procedures, by personnel equipped with powered air-purifying respirators.
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5

SARS-CoV-2 Propagation and Concentration

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The USA reference strain SARS-CoV-2 USA-WA 1/2020 (BEI resources NR-52281) was propagated in African green monkey kidney cells (Vero-E6 ATCC CRL-1586) as described previously [36 (link)]. Handling of SARS-CoV-2 was carried out under strict BSL3 biosafety protocols at the Center for Food Safety BSL3 laboratory. Briefly, one- or two-day-old 90% confluent cells were used to prepare virus stocks using a multiplicity of infection of 0.01. Infection media consisted of DMEM supplemented with 2% heat-inactivated fetal bovine serum and 1% antibiotic–antimycotic cocktail. Harvesting the virus was carried out at 72 h post-infection. Infected cells were collected from the flasks and centrifuged at 450× g for 5 min at 4 °C to pellet the cell debris, while supernatants containing the virus were ultrafiltered through Amicon 100 KDa Ultra-15 centrifugal devices (MilliporeSigma, St. Louis, MO, USA) immediately after harvest to concentrate the virus 10 times, while semi-purifying it from cell culture lysates [37 (link)]. The latter would also overcome the buffering effect of infection media in virus stocks on NaOCl. An aliquot of the virus was immediately titrated as described below. The original viral titer generated was ~7 log10 TCID50/mL, while the ultrafiltered virus titer was ~8 log10 TCID50/mL.
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6

Cell Culture Conditions for HEK293T, HeLa, and VeroE6 Cells

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Human HEK293T (American Type Culture Collection [ATCC]), HeLa (NIH HIV Reagent Program, Division of AIDS, NIAID, NIH from Dr. Richard Axel), HEK293T-ACE2 (BEI resources), and VeroE6 (ATCC) cells were cultured in complete medium (Dulbecco’s Modified Eagle Medium [DMEM, ThermoFisher Scientific] supplemented with 10% fetal bovine serum [FBS, ThermoFisher Scientific], 1% Penicillin-Streptomycin [ThermoFisher Scientific] and 1% L-glutamine [ThermoFisher Scientific]). A549-ACE2 (BEI resources) cells were maintained in complete medium with 50 ng/mL blasticidin S HCl (ThermoFisher Scientific). HEK293T-ACE2 stably expressing pQCXIP-BST2 or pQCXIP, HEK293T-ACE2-BST2-ATG5KO and HEK293T-ACE2-BST2-LentiCRISPRv2 cells were grown in complete medium with 1 μg/mL of puromycin (ThermoFisher Scientific). A549-ACE2 cells stably expressing pQCXIP-BST2 or pQCXIP were maintained in complete medium with 50 ng/mL blasticidin S HCl and 1 μg/mL of puromycin. Viability of cells was measured after each transfection. Only cells with viabilities > 90% were considered for further experiments.
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7

SARS-CoV-2 Propagation in Vero E6 Cells

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Vero cells (CCL-81) were obtained originally from the International Reagent Resource and frozen stocks prepared. Those stocks screened negative for mycoplasma contamination and were used between total passage level 35 and 41. Virus (isolate USA-WA1/2020) was acquired through BEI Resources (product NR-52281) and amplified in Vero C1008 (Vero E6) cell culture. Vero E6 cells (ATCC CRL-1568) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with glucose, L-glutamine, sodium pyruvate, 5% fetal bovine serum (FBS) and antibiotics. Inoculation of Vero E6 cells with SARS-CoV-2 was carried out directly in DMEM containing (1%) FBS. The identity of the virus as SARS-CoV-2 was established by shotgun sequencing and confirmation with a database on sequence data for the virus. Medium harvested from infected cells 3–4 days after inoculation was clarified by centrifugation, supplemented with FBS to 10% and frozen to -80°C in aliquots. All virus propagation occurred in a BSL-3 laboratory setting. We measured all values for virus concentration using a standard plaque assay. Results are presented in number of infectious virus (plaque forming units per mL).
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8

SARS-CoV-2 Propagation in Vero E6 Cells

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VeroE6 (CRL-1586, ATCC®) was used for infection experiments. Cells were cultured in complete media prepared using Dulbecco's modified Eagle's medium (DMEM; 12100–038, Gibco) supplemented with 10% HI-FBS (16140–071, Gibco), 100 U/mL Penicillin-Streptomycin (15140122, Gibco) and GlutaMAX™ (35050–061, Gibco). SARS-CoV-2 (Isolate Hong Kong/VM20001061/2020, NR-52282, BEI Resources, NIAID, NIH) was propagated and titrated by plaque assay in Vero E6 cells as described in the literature [17 (link)].
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9

SARS-CoV-2 Amplification in Vero E6 Cells

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Vero E6 (CRL-1586; American Type Culture Collection) were cultured Dulbecco’s Modified Eagle’s Medium (DMEM,Corning) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologics) and 1% nonessential amino acids (NEAA,Corning). SARS-CoV-2, isolate USA-WA1/2020 19 (BEI resources #NR52281), a gift from Dr. Mark Mulligan at the NYU Langone Vaccine Center was amplified once in Vero E6cells. All experiments with SARS-CoV-2 were conducted in the NYU Grossman School of Medicine ABSL3 facility by personnel equipped with powered air-purifying respirators.
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10

SARS-CoV-2 Virus Variant Propagation and Pseudotyping

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Vero E6 (CRL-1586, American Type Culture Collection (ATCC) and 293T (ATCC) were cultured at 37°C in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES pH 7.3, 1 mM sodium pyruvate, and 100 U/mL of penicillin–streptomycin. 293T was cultured in DMEM medium. 293T-Spike and 293T-ACE2 were cultured in DMEM medium containing 100 μg/mL Zeocin. Expi293F was maintained in Expi293™ Expression Medium (ThermoFisher, Cat# A1435103). The SARS-CoV-2 spike pseudotyped HIV-1 backboned virus were packaged in 293T cells after transfecting pNL4-3.luc.RE and pcDNA3.1-spike plasmids (WT, Alpha, Beta, Gamma, Delta, Omicron BA.1 and Omicron B.2). The SARS-CoV-2 live virus variants (WT, Alpha, Beta, Delta, Omicron BA.1) ordered from BEI Resources and propagated VeroE6 cells. The mouse ACE2 adapted SAR-CoV-2 virus (Beta variants) gene recovered by the reverse genetics was produced in VeroE6 cells. All work with infectious SARS-CoV-2 was performed in Institutional Biosafety Committee approved BSL3 facilities using appropriate positive pressure air respirators and protective equipment.
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