Mdck cells

Manufactured by American Type Culture Collection

Sourced in United States, Germany, United Kingdom

MDCK cells are a well-established cell line derived from the kidney of a normal adult female cocker spaniel. These cells are commonly used in research to study various biological processes, including cell signaling, protein trafficking, and virus-host interactions. The MDCK cell line maintains a stable epithelial phenotype and is a widely utilized model system for studying cell polarity and tight junction formation.

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MDCK cell line (16 citations) MDCK II cells (7 citations) MDCK CCL-34) cells (6 citations) MDCK II canine kidney epithelial cells (3 citations) Madin-Darby canine kidney cells (MDCK) (3 citations) MDCK) cells line (2 citations) MDCK canine kidney cells (2 citations) MDCK and A549 human lung epithelial cells (2 citations) MDCK renal cells (2 citations) MDCK) II cell lines (2 citations)

175 protocols using mdck cells

Generation of H9N2 Influenza Virus

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Recombinant A/chicken/Pakistan/UDL01/08 H9N2 virus was generated using reverse genetics as previously described [66 (link)]. Virus stocks were produced via passage in 10 day old embryonated chicken eggs; the allantoic fluid harvested after 48 h and titrated by plaque assay on MDCK cells (ATCC).
Madin-Darby Canine Kidney (MDCK) cells (ATCC) were maintained in DMEM (Gibco-Invitrogen, Inc.) supplemented with 10% foetal bovine serum (Biosera, Inc.), 1% penicillin/streptomycin (Sigma-Aldrich, Inc.) and 1% non-essential aa (Sigma-Aldrich, Inc.).

Chrzastek K., Leng J., Zakaria M.K., Bialy D., La Ragione R, & Shelton H. (2021). Low pathogenic avian influenza virus infection retards colon microbiota diversification in two different chicken lines. Animal Microbiome, 3, 64.

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Propagation and Titration of H7N9 Influenza Virus

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A/Shanghai/2/2013 (H7N9)-PR8-IDCDC-RG32A (Influenza Reagent Resource) were propagated and titered in monolayer cultures of MDCK cells (ATCC). Cells were maintained in Dulbecco’s Modified Eagle Medium (GIBCO DMEM, Invitrogen) at 37°C in 5% CO₂. H7N9 A/Shanghai/2/2013 (H7N9)-PR8-IDCDC-RG32A were manipulated under BSL-2 conditions with BSL-3 practices.

Gilchuk I.M., Bangaru S., Gilchuk P., Irving R.P., Kose N., Bombardi R.G., Thornburg N.J., Creech C.B., Edwards K.M., Li S., Turner H.L., Yu W., Zhu X., Wilson I.A., Ward A.B, & Crowe JE J.r. (2019). Influenza H7N9 Virus Neuraminidase-Specific Human Monoclonal Antibodies Inhibit Viral Egress and Protect from Lethal Influenza Infection in Mice. Cell host & microbe, 26(6), 715-728.e8.

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Influenza Virus Propagation and Titration

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Influenza A/Puerto Rico/8/1934 (H1N1) (A/PR/8/34) virus was obtained from the American Type Culture Collection (ATCC). Influenza A/Chicken/México/31381-7/1994 (H5N2) was donated by A. Aguilar Setien. Madin-Darby canine kidney (MDCK) cells (ATCC, Manassas, VA, UAS) were maintained in Dubelco Eagle’s Minimum Essential Medium supplemented with 5% fetal bovine serum and a mixture of antibiotics and antimycotics (100 U of penicillin, 0.1 mg of streptomycin, and 0.25 μg of amphotericin B/mL). The supernatants of the infected cultures were obtained after 96 h post-infection, clarified by centrifugation at low speed and stored at −70 °C for further use. Viral inoculum was titrated through the formation of lytic plaques in the same cell line using the conventional method in medium solidified with 0.5% agarose and stained with violet crystal [69 (link)]. Titers were reported in plaque forming units per milliliter (PFU/mL).

Márquez-Domínguez L., Reyes-Leyva J., Herrera-Camacho I., Santos-López G, & Scior T. (2020). Five Novel Non-Sialic Acid-Like Scaffolds Inhibit In Vitro H1N1 and H5N2 Neuraminidase Activity of Influenza a Virus. Molecules, 25(18), 4248.

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Antiviral Efficacy of PPa Against Influenza Strains

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MDCK cells (ATCC) were cultured in 96-wellg plates (2 × 10⁴ cells/well) for 24 h. A series of double-diluted PPa solutions was pre-incubated with 100 TCID₅₀ of the virus at 37 °C for 30 min, and these cells were incubated with a virus-compound mixture for 1 h after two washes with PBS. Then, 1 μg/mL of TPCK-trypsin (trypsin treated with TPCK, Sigma, USA) in serum-free DMEM was added to the cells. Next, cell viability was measured using the MTT method at 48 h after the infection. S-KKWK [17] (link) was used as a positive control, and the experiment was independently repeated at least three times. Virus subtypes such as the A/FM-1/1/47 (H1N1) mouse-adapted strain, A/Puerto Rico/8/34 (H1N1), A/Puerto Rico/8/34 (H1N1) with the NA-H274Y mutation, and A/Aichi/2/68 (H3N2) were selected to evaluate the antiviral effects of PPa.

Chen D., Lu S., Yang G., Pan X., Fan S., Xie X., Chen Q., Li F., Li Z., Wu S, & He J. (2020). The seafood Musculus senhousei shows anti-influenza A virus activity by targeting virion envelope lipids. Biochemical Pharmacology, 177, 113982.

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Cell Line Maintenance Protocols

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HeLa, HEK 293 T and MT4 cells were described before³⁴. MDCK cells were obtained from ATCC and maintained in DMEM supplemented with 10% FBS. Murine myeloma NSO cells were from ATCC and maintained in RPMI 1640 supplemented with 2 mM glutamine and 10% FBS. MEF cells were a kind gift from Dr. Farzad Mahjour (University of Isfahan). S enterica and E. coli were kindly provided by Dr. Hamid-Reza Khojasteh (University of Isfahan).

Romani B., Kavyanifard A, & Allahbakhshi E. (2017). Antibody production by in vivo RNA transfection. Scientific Reports, 7, 10863.

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MDCK Cell Culture and Harvesting

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MDCK cells (ATCC) at 80% confluency where trypsinized for 12 minutes,
then 4 ml of DMEM (Genesee) containing 10% FBS (GIBCO) was added to neutralize
the trypsin. That volume was then spun down at 200 RPM for 5 minutes, the media
decanted, and then resuspended in 1ml of PBS (Fisher) containing 3% FBS.

Noren B.E., Shaha R.K., Stenquist A.T., Frick C.P, & Oakey J.S. (2019). Cell Printing in Complex Hydrogel Scaffolds. IEEE transactions on nanobioscience, 18(2), 265-268.

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Influenza Virus Infection in Ferrets

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Madin‐Darby canine kidney cells (MDCK cells, ATCC) were maintained in Eagle's minimum essential media (Wisent), 10% fetal bovine serum (FBS, Wisent), and penicillin (100 UI/mL)/streptomycin (100 μg/mL; P/S, Wisent). Influenza A/Ontario/2016 (H1N1)‐like and influenza A/Ontario/2016 (H3N2)‐like viruses were obtained from clinical samples at Sunnybrook Health Sciences Centre (Toronto, Canada) and subsequently passaged and plaque‐purified.35 These were used for artificial aerosolization experiments. Ferrets were inoculated with egg‐passaged influenza A/California/07/2009 virus (Cal/09).

Bekking C., Yip L., Groulx N., Doggett N., Finn M, & Mubareka S. (2019). Evaluation of bioaerosol samplers for the detection and quantification of influenza virus from artificial aerosols and influenza virus–infected ferrets. Influenza and Other Respiratory Viruses, 13(6), 564-573.

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Virus Titration via ELISA and EID50 Assays

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ELISA (enzyme-linked immunosorbent assay) was used to measure TCID₅₀ titers as previously reported [19 (link)], MDCK cells (ATCC, Manassas, VA, USA) were grown on 96-well flat-bottom microplates at 37 °C with 5% CO₂. MDCK cells with 80–90% confluence were washed with 1× concentrated PBS and then inoculated with serial 10-fold dilutions of virus suspensions in media containing 1 µg/mL of TPCK-trypsin. Virus-infected cells were incubated at 37 °C with 5% CO₂ for 72 h. Next, the TCID₅₀ titers were determined via the Reed and Muench method [20 (link)]. To determine EID₅₀, the allantoic cavities of 10-day-old SPF ECEs were inoculated with 100 μL serial 10-fold dilutions of the viruses, using 5 eggs for each dilution. The eggs were incubated at 37 °C for 96 h. Allantoic fluid was harvested and tested using HA assays [21 ], and EID₅₀ calculation of viruses was completed using the Reed and Muench method [20 (link)].

Durairaj K., Trinh T.T., Yun S.Y., Yeo S.J., Sung H.W, & Park H. (2022). Molecular Characterization and Pathogenesis of H6N6 Low Pathogenic Avian Influenza Viruses Isolated from Mallard Ducks (Anas platyrhynchos) in South Korea. Viruses, 14(5), 1001.

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Culturing MDCK and HEK293T cells

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MDCK cells (ATCC) were cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% FBS, L-glutamine and penicillin/streptomycin at 37°C in a 5% CO₂ atmosphere. HEK293T cells (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS, L-glutamine and penicillin/streptomycin at 37°C in a 5% CO₂ atmosphere.

Le Sage V., Kanarek J.P., Snyder D.J., Cooper V.S., Lakdawala S.S, & Lee N. (2020). Mapping of Influenza Virus RNA-RNA Interactions Reveals a Flexible Network. Cell reports, 31(13), 107823.

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Propagation and Inactivation of Pandemic Swine Flu Virus

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A strain of 2009 pandemic swine flu (H1N1 A/Auckland/3/2009) was obtained from the World Health Organization (WHO Melbourne) in 2010. Viral stocks were propagated in Madin-Darby Canine Kidney (MDCK) cells (ATCC, Manassas, VA, USA), as previously described[17 (link)–19 (link)]. Stock viral concentrations were measured using plaque assays; which determines live virions based on plaque forming units per ml (pfu/ml)[17 (link)–19 (link)]. Ultra-violet (UV) inactivation of live viruses was achieved by placing live viruses directly under a UV lamp (254nm) for 3hrs. Successful inactivation was confirmed by plaque assays.

Tolosa J.M., Parsons K.S., Hansbro P.M., Smith R, & Wark P.A. (2015). The Placental Protein Syncytin-1 Impairs Antiviral Responses and Exaggerates Inflammatory Responses to Influenza. PLoS ONE, 10(4), e0118629.

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