Vero ccl81

Manufactured by American Type Culture Collection

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The Vero CCL81 is a cell line derived from the kidney of an African green monkey (Cercopithecus aethiops). It is a widely used cell line for various applications in cell biology and virology research.

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Vero cell line (ATCC-CCL81), (2 citations) Vero (No CCL81) (2 citations)

51 protocols using vero ccl81

Cell Lines and Viral Strains for PEDV Research

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Vero CCL-81 (ATCC), Huh7 (Procell, Wuhan, China), HepG2 (Procell), Hep3B217 (known as Hep 3B2.1-7, Procell), and BHK21 (Procell) were cultured in DMEM (Hyclone, Shanghai, China) containing 10% fetal bovine serum (FBS) (Gibco, Shanghai, China) and 1% penicillin/streptomycin (Gibco). Li-7 (Procell), SNU182 (MeiSenCTCC, Hangzhou, China), SNU387 (MeiSenCTCC), and SNU761 (MeiSenCTCC) were cultured in RPMI 1640 (Hyclone, Shanghai, China) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco). The PEDV-SD strain was isolated and stored in our laboratory. Recombinant rPEDV-EGFP was produced by transfecting infectious cDNA clones into BHK21 cells as previously described [16 (link)]. The viruses were titrated on the Vero CCL-81 cells by TCID₅₀. Rabbit monoclonal antibody against PEDV N was purchased from Shanghai Ango Biotech.

Lv L., Luo H., Yu L., Tong W., Jiang Y., Li G., Tong G., Li Y, & Liu C. (2022). Identification and Characterization of Cell Lines HepG2, Hep3B217 and SNU387 as Models for Porcine Epidemic Diarrhea Coronavirus Infection. Viruses, 14(12), 2754.

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SARS-CoV-2 Infection of Engineered Vero Cells

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Vero E6 (CRL-1586, American Type Culture Collection (ATCC), Vero CCL81 (ATCC), Vero-furin (Mukherjee et al., 2016 (link)), and Vero-hACE2-TMPRSS2 (a gift of A. Creanga and B. Graham, NIH) 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, 1× non-essential amino acids, and 100 U/ml of penicillin–streptomycin. Additionally, Vero-hACE2-TMPRSS2 cells were cultured in the presence of 5 μg/mL puromycin. The WA1/202 (2019n-CoV/USA_WA1/2020) isolate of SARS-CoV-2 was obtained from the US Centers for Disease Control (CDC). The B.1.1.7 and WA1/2020 E484K/N501Y/D614G viruses have been described previously (Chen et al., 2021 ; Xie et al., 2021a ). Infectious stocks were propagated by inoculating Vero CCL81 or Vero-hACE2-TMPRSS2 cells. Supernatant was collected, aliquoted, and stored at −80°C. All work with infectious SARS-CoV-2 was performed in Institutional Biosafety Committee-approved BSL3 and A-BSL3 facilities at Washington University School of Medicine using positive pressure air respirators and protective equipment.

Case J.B., Chen R.E., Cao L., Ying B., Winkler E.S., Goreshnik I., Shrihari S., Kafai N.M., Bailey A.L., Xie X., Shi P.Y., Ravichandran R., Carter L., Stewart L., Baker D, & Diamond M.S. (2021). Ultrapotent miniproteins targeting the receptor-binding domain protect against SARS-CoV-2 infection and disease in mice. bioRxiv.

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SARS-CoV-2 Variant Propagation in Engineered Vero Cells

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Vero E6 (CRL-1586, American Type Culture Collection (ATCC), Vero CCL81 (ATCC), Vero-furin (Mukherjee et al., 2016 (link)), and Vero-hACE2-TMPRSS2 (a gift of A. Creanga and B. Graham, NIH) 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, 1 × non-essential amino acids, and 100 U/mL of penicillin–streptomycin. Additionally, Vero-TMPRSS2 and Vero-hACE2-TMPRSS2 cells were cultured in the presence of 5 μg/mL of blasticidin or puromycin, respectively. The WA1/202 (2019n-CoV/USA_WA1/2020) isolate of SARS-CoV-2 was obtained from the US Centers for Disease Control (CDC). The B.1.1.7, WA1/2020-N501Y/D614G, and WA1/2020-E484K/N501Y/D614G viruses have been described previously (Chen et al., 2021 (link); Xie et al., 2021 (link)). Infectious stocks were propagated by inoculating Vero CCL81 or Vero-hACE2-TMPRSS2 cells. Supernatant was collected, aliquoted, and stored at −80°C. All work with infectious SARS-CoV-2 was performed in Institutional Biosafety Committee-approved BSL3 and A-BSL3 facilities at Washington University School of Medicine using positive pressure air respirators and protective equipment. All virus stocks were deep-sequenced after RNA extraction to confirm the presence of the anticipated substitutions.

Case J.B., Chen R.E., Cao L., Ying B., Winkler E.S., Johnson M., Goreshnik I., Pham M.N., Shrihari S., Kafai N.M., Bailey A.L., Xie X., Shi P.Y., Ravichandran R., Carter L., Stewart L., Baker D, & Diamond M.S. (2021). Ultrapotent miniproteins targeting the SARS-CoV-2 receptor-binding domain protect against infection and disease. Cell Host & Microbe, 29(7), 1151-1161.e5.

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SARS-CoV-2 Virus Propagation in Vero Cells

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Vero E6 (CRL-1586, American Type Culture Collection (ATCC), Vero CCL81 (ATCC), and Vero-furin cells⁴⁷ 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, 1× non-essential amino acids, and 100 U/ml of penicillin–streptomycin. The 2019n-CoV/USA_WA1/2019 isolate of SARS-CoV-2 was obtained from the US Centers for Disease Control (CDC). Infectious stocks were grown by inoculating Vero CCL81 cells and collecting supernatant upon observation of cytopathic effect; debris was removed by centrifugation and passage through a 0.22 μm filter. Supernatant was then aliquoted and stored at −80°C.

Winkler E.S., Bailey A.L., Kafai N.M., Nair S., McCune B.T., Yu J., Fox J.M., Chen R.E., Earnest J.T., Keeler S.P., Ritter J.H., Kang L.I., Dort S., Robichaud A., Head R., Holtzman M.J, & Diamond M.S. (2020). SARS-CoV-2 infection of hACE2 transgenic mice causes severe lung inflammation and impaired function. Nature immunology, 21(11), 1327-1335.

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Propagation and Quantification of HSV Variants

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HSV1/TF+ and HSV/TF− were propagated in TF-inducible human melanoma cells [24 (link)] and purified as previously described [23 (link)]. Comparable in vitro plaque forming units (PFU) per virus particle (VP) number was ensured [25 (link)]. Thus, each virus preparation had similar numbers of VP/mL and PFU/mL. This was achieved by introducing UV-inactivated virus into higher PFU/VP preparations. Viruses were purified by sucrose gradient ultracentrifugation, quality assured for lack of cellular debris and quantified to derive VP/mL by negative staining electron microscopy [25 (link)]. All cells were shown to be mycoplasma free. To derive the number of infectious viruses, plaque assays on African green monkey kidneys cells (Vero, CCL-81; ATCC, Manassas, VA) were conducted, as previously described [23 (link)].

Sutherland M.R., Simon A.Y., Shanina I., Horwitz M.S., Ruf W, & Pryzdial E.L. (2019). Virus envelope tissue factor promotes infection in mice. Journal of thrombosis and haemostasis : JTH, 17(3), 482-491.

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Culturing HEK293T, Vero, and Mouse Splenocytes

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Human embryonic kidney 293T (HEK293T; ATCC-CLR-N268) and Vero CCL-81 (ATCC #CCL-81) (ATCC, Manassas, VA, USA) cells were cultured in D10 media: Dulbecco Modified Eagle's Medium (Invitrogen Life Science Technologies, San Diego, CA, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS), 3 mM glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin [23 (link)]. Mouse splenocytes were cultured in R10 media: (RPMI1640, Invitrogen Life Science Technologies, San Diego, CA, USA) supplemented with 10% heat-inactivated FCS, 3 mM glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin. All cell types were cultured in incubators set to 37°C and 5% CO₂.

Choi H., Kudchodkar S.B., Reuschel E.L., Asija K., Borole P., Ho M., Wojtak K., Reed C., Ramos S., Bopp N.E., Aguilar P.V., Weaver S.C., Kim J.J., Humeau L., Tebas P., Weiner D.B, & Muthumani K. (2019). Protective immunity by an engineered DNA vaccine for Mayaro virus. PLoS Neglected Tropical Diseases, 13(2), e0007042.

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SARS-CoV-2 Infection Protocols in Cell Lines

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Vero E6 (CRL-1586, American Type Culture Collection (ATCC), Vero CCL81 (ATCC), and HEK293 cells 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, 1X non-essential amino acids, and 100 U/ml of penicillin–streptomycin
SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020 was obtained from the Centers for Disease Control and Prevention (a gift from Natalie Thornburg). The virus was passaged once in Vero CCL81 cells and titrated by focus-forming assay (FFA) on Vero E6 cells. The recombinant luciferase-expressing full-length SARS-CoV-2 reporter virus (2019 n-CoV/USA_WA1/2020 strain) has been reported previously (Zost et al., 2020 (link)), and the D614G variant will be described elsewhere (R. Baric, manuscript in preparation). All work with infectious SARS-CoV-2 was performed in Institutional Biosafety Committee approved BSL3 and A-BSL3 facilities using appropriate positive pressure air respirators and protective equipment.

Hassan A.O., Kafai N.M., Dmitriev I.P., Fox J.M., Smith B.K., Harvey I.B., Chen R.E., Winkler E.S., Wessel A.W., Case J.B., Kashentseva E., McCune B.T., Bailey A.L., Zhao H., VanBlargan L.A., Dai Y.N., Ma M., Adams L.J., Shrihari S., Danis J.E., Gralinski L.E., Hou Y.J., Schäfer A., Kim A.S., Keeler S.P., Weiskopf D., Baric R.S., Holtzman M.J., Fremont D.H., Curiel D.T, & Diamond M.S. (2020). A Single-Dose Intranasal ChAd Vaccine Protects Upper and Lower Respiratory Tracts against SARS-CoV-2. Cell, 183(1), 169-184.e13.

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Cultivation of Diverse Cell Lines

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Vero E6 (CRL-1586, American Type Culture [ATCC]), Vero CCL81 (CCL-81, ATCC), and HEK293T (CRL-3216 ATCC) were maintained at 37°C in 5% CO₂ in Dulbecco’s minimal essential medium (DMEM) containing 10% (vol/vol) heat-inactivated fetal bovine serum (FBS), 10 mM HEPES pH 7.3, 1 mM sodium pyruvate, 1× non-essential amino acids, and 100 U/mL of penicillin-streptomycin. Vero-furin cells were obtained from T. Pierson (NIH) and have been described previously³⁹. Expi293F cells (ThermoFisher Scientific, A1452) were maintained at 37°C in 8% CO₂ in Expi293F Expression Medium (ThermoFisher Scientific, A1435102). ExpiCHO cells (ThermoFisher Scientific, A29127) were maintained at 37°C in 8% CO₂ in ExpiCHO Expression Medium (ThermoFisher Scientific, A2910002). Mycoplasma testing of Expi293F and ExpiCHO cultures was performed on a monthly basis using a PCR-based mycoplasma detection kit (ATCC, 30-1012K).

Zost S.J., Gilchuk P., Chen R.E., Case J.B., Reidy J.X., Trivette A., Nargi R.S., Sutton R.E., Suryadevara N., Chen E.C., Binshtein E., Shrihari S., Ostrowski M., Chu H.Y., Didier J.E., MacRenaris K.W., Jones T., Day S., Myers L., Lee F.E., Nguyen D.C., Sanz I., Martinez D.R., Rothlauf P.W., Bloyet L.M., Whelan S.P., Baric R.S., Thackray L.B., Diamond M.S., Carnahan R.H, & Crowe JE J.r. (2020). Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein. Nature medicine, 26(9), 1422-1427.

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Cell Culture and RSV Propagation

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Human epidermoid cancer cell line (HEp-2, CCL-23) and African green monkey kidney epithelial cell line (Vero, CCL-81), originally obtained from ATCC (Manassas, VA, USA), were purchased from Cell Bank of Chinese Academy Sciences (Shanghai, China). Human lung adenocarcinoma epithelial cell line (A549, CCL-185) was purchased from the ATCC. The cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium, high glucose, Life Technologies, Carlsbad, CA, USA), supplemented with 10% heat inactivated fetal bovine serum (FBS), L-glutamine, nonessential amino acids, and sodium pyruvate at 37 °C in a humidified atmosphere with 5% CO₂. Human RSV strain A2 was propagated in A549 cells as previously described45 (link). Because RSV retains a close association with the host cell membrane and has a tendency to aggregate during centrifugation procedures, the stock of crude supernatant was used after a titer was determined.

Shi H., Ren K., Lv B., Zhang W., Zhao Y., Tan R.X, & Li E. (2016). Baicalin from Scutellaria baicalensis blocks respiratory syncytial virus (RSV) infection and reduces inflammatory cell infiltration and lung injury in mice. Scientific Reports, 6, 35851.

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Visualizing SARS-CoV-2 and Influenza Antigens

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Vero (CCL-81, ATCC, Manassas, VA, USA) and MDCK (ECAAC 84121903, Sigma-Aldrich, St. Louis, MO, USA) cells infected with SARS-CoV-2 M2SR, Sing2016 H3N2 M2SR (MOI = 5) or media (mock) were fixed at 24 h post-infection with 4% formalin, and then incubated with the human anti-SARS-CoV RBD monoclonal CR3022 (Abcam, Boston, MA, USA) or mouse anti-H3 HA monoclonal (NR-52364, BEI, Manassas, VA, USA) antibody, followed by secondary antibody goat anti-human IgG Alexa Fluor 488 or goat anti-mouse IgG Alexa Fluor 647 conjugates (Southern Biotech, Birmingham, AL, USA). Labeled cells were stained with DAPI and visualized using an ECHO fluorescent microscope on FITC, Cy5 and DAPI channels, respectively.

Moser M.J., Hill-Batorski L., Bowen R.A., Matejka S.M., Marshall D., Kawaoka Y., Neumann G, & Bilsel P. (2023). Intranasal Single-Replication Influenza Vector Induces Cross-Reactive Serum and Mucosal Antibodies against SARS-CoV-2 Variants. Vaccines, 11(6), 1063.

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