Embryonated chicken eggs

Manufactured by Charles River Laboratories

Sourced in United States

Embryonated chicken eggs are a commonly used laboratory resource. They serve as a substrate for the cultivation and propagation of various organisms, including viruses and other microbiological agents. The eggs provide a natural, nutrient-rich environment that supports the growth and development of these biological entities. Embryonated chicken eggs are a versatile and widely utilized tool in various research and testing applications.

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37 protocols using embryonated chicken eggs

Generation of Recombinant Influenza Viruses

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PR8-miRctrl and PR8-miR133b/206 were generated as previously described (38 (link)–44 (link)). Briefly, two copies each of target sequences for miR133b and miR206, or a length-matched untargeted sequence, were cloned and ligated into the 3′ untranslated region of the NP gene along with a duplicated 3′ NP packaging sequence. The recombinant viruses with modified NP segments were rescued using reverse genetic techniques and plaque-purified. Viruses were propagated in 10-day-old embryonated chicken eggs (Charles River Laboratories) for 48 hours at 37°C and titered on Madin-Darby canine kidney (MDCK) cells. For determining organ titers, tissues were collected and homogenized in 500 μl of phosphate-buffered saline (PBS), flash-frozen, and stored at −80°C before titering on MDCK cells.

Kenney A.D., Aron S.L., Gilbert C., Kumar N., Chen P., Eddy A., Zhang L., Zani A., Vargas-Maldonado N., Speaks S., Kawahara J., Denz P.J., Dorn L., Accornero F., Ma J., Zhu H., Rajaram M.V., Cai C., Langlois R.A, & Yount J.S. (2022). Influenza virus replication in cardiomyocytes drives heart dysfunction and fibrosis. Science Advances, 8(19), eabm5371.

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Mice Infection Protocol in Germany

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All experiments with mice were approved by an external committee according to the national guidelines of the animal welfare law in Germany (BGBl. I S. 1206, 1313 and BGBl. I S. 1934). The protocol used in these experiments has been reviewed by an ethics committee and approved by the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany (permit number 3392 42502-04-13/1234). Mice were maintained under specific-pathogen-free conditions at the animal facilities of the Helmholtz Centre for Infection Research (HZI). Embryonated chicken eggs were purchased from Charles River Laboratories, Germany.

Kühn N., Bergmann S., Kösterke N., Lambertz R.L., Keppner A., van den Brand J.M., Pöhlmann S., Weiß S., Hummler E., Hatesuer B, & Schughart K. (2016). The Proteolytic Activation of (H3N2) Influenza A Virus Hemagglutinin Is Facilitated by Different Type II Transmembrane Serine Proteases. Journal of Virology, 90(9), 4298-4307.

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Baculovirus Rescue and Influenza A Virus Propagation

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Sf9 cells (CRL-1711, ATCC) for baculovirus rescue transfection were grown in Trichoplusia ni Medium-Formulation Hink (TNM-FH) insect cell medium (Gemini Bioproducts) supplemented with 10% fetal bovine serum (FBS) (Sigma) and penicillin (100 U/ml)/streptomycin (100 μg/ml) (P/S) (Gibco) solution. BTI-TN-5B1-4 (High Five) cells for protein expression were grown in serum-free SFX-insect medium (HyClone) supplemented with P/S solution. Madin Darby canine kidney (MDCK) cells, human embryonic kidney 293 (HEK293) cells, tetracycline repressor (T-Rex)-293 cell line (Thermo Fisher) and the DF-1 chicken embyo fibroblast cell line were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% FBS and P/S solution. The influenza A virus strain, A/Wyoming/03/2003 (H3N2), was grown in 10-days-old embryonated chicken eggs (Charles River) for 48 h at 37°C. Eggs were then cooled overnight at 4°C, before the allantoic fluid was harvested the next day. Harvested allantoic fluid was centrifuged at 4,000 × g for 10 min at 4°C to remove debris. Viruses were then aliquoted and stored at −80°C.

McMahon M., Asthagiri Arunkumar G., Liu W.C., Stadlbauer D., Albrecht R.A., Pavot V., Aramouni M., Lambe T., Gilbert S.C, & Krammer F. (2019). Vaccination With Viral Vectors Expressing Chimeric Hemagglutinin, NP and M1 Antigens Protects Ferrets Against Influenza Virus Challenge. Frontiers in Immunology, 10, 2005.

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Propagation and Characterization of Influenza Strains

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The influenza A virus strains A/Singapore/INFIMH-16-0019/2016 IVR-186 (H3N2), A/Switzerland/9715293/2013 (H3N2) (mouse-adapted), A/Philippines/2/1982 (H3N2, X-79, A/PR/8/1934 reassortant), A/duck/Czechoslovakia/1956 (H4N6, A/PR/8/1934 reassortant) (52 (link)), A/Shanghai/1/2013 (H7N9, A/PR/8/1934 reassortant), A/canine/Illinois/41915/2015 (H3N2) and A/Netherlands/602/09 (H1N1) were grown in 10-d-old embryonated chicken eggs (Charles River) for 48 h at 37 °C. After virus growth, the eggs were chilled at 4 °C overnight. The following day the allantoic fluid was harvested from the chilled eggs and cellular debris was removed via centrifugation (4,000 × g for 10 min at 4 °C). Viruses were aliquoted and stored at −80 °C. Virus titers (plaque forming units/mL) were determined via plaque assay.
A/Singapore/INFIMH-16-0019/2016 IVR-186 (H3N2), A/Philippines/2/1982 (H3N2, X-79), A/duck/Czechoslovakia/1956 (H4N6) and A/Shanghai/1/2013 (H7N9) are reassortant viruses and contain the internal genome segments of A/Puerto Rico/8/1934 (H1N1).

McMahon M., O’Dell G., Tan J., Sárközy A., Vadovics M., Carreño J.M., Puente-Massaguer E., Muramatsu H., Bajusz C., Rijnink W., Beattie M., Tam Y.K., Kirkpatrick Roubidoux E., Francisco I., Strohmeier S., Kanekiyo M., Graham B.S., Krammer F, & Pardi N. (2022). Assessment of a quadrivalent nucleoside-modified mRNA vaccine that protects against group 2 influenza viruses. Proceedings of the National Academy of Sciences of the United States of America, 119(45), e2206333119.

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Influenza Virus Propagation in Cell Lines

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Embryonated chicken eggs were purchased from Charles River Laboratories (Willimantic, CT) and then incubated at 37.5 °C for up to 9 days for use in the propagation of engineered influenza viruses. MDCK cells (ATCC, cat. no. CCL-34) were cultured in Eagle’s minimum essential medium (EMEM; ATCC, cat. no. 30–2003) supplemented with 10% FBS (Gibco, NY). 293T cells (ATCC, cat. no. CRL-11268), MEF cells (ATCC, cat. no. CRL-2214), and A549 cells (ATCC, cat. no. CCL-185) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, NY) supplemented with 10% FBS, 1% penicillin, and 1 μg/ml streptomycin (Gibco, NY). Cells were cultured at 37°C in 5% CO₂.

Wen K., Wang H., Chen Y., Yang H., Zheng Z., Yan Y., Adilene R, & Zeng M. (2021). A new self-attenuated therapeutic influenza vaccine that uses host cell-restricted attenuation by artificial microRNAs. International journal of pharmaceutics, 612, 121325.

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Murine Influenza Model with IFN-γ Knockout

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BALB/c and C57Bl/6 IFN-γ^+/+ and IFN-γ ^−/− mice were originally obtained from Jackson Laboratories (Bar Harbor, ME). 3Rora^+/floxIl7r^Cre mice were generated in the lab of Dr. Andrew N.J. McKenzie (MRC Laboratory of Molecular Biology) ^{24 (link)}. All mice were maintained under specific pathogen-free conditions in the Animal Research Facility at Albany Medical College. Mice were anaesthetized by intraperitoneal (i.p.) injection with 100 μl xylazine (20 mg/ml) and ketamine (1 mg/ml) in PBS. For influenza infections, anaesthetized IFN-γ^+/+ and IFN-γ ^−/− mice were intranasally (i.n.) inoculated with a lethal dose (2000 PFU) H1N1 A/California/04/2009 (CA04). ILC2 deficient animals and their littermate controls (Rora^+/floxIl7r^Cre) were given the median lethal dose (LD₅₀) of 500 PFU. Survival and weight loss were monitored daily for 20 days. The virus was propagated in 10-day-old embryonated chicken eggs (Charles River). The stock titer was measured by standard plaque assay. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Albany Medical College (Protocol Number 11-04004).

Califano D., Furuya Y., Roberts S., Avram D., McKenzie A.N, & Metzger D.W. (2017). IFN-γ increases susceptibility to influenza A infection through suppression of group II innate lymphoid cells. Mucosal immunology, 11(1), 209-219.

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Influenza Virus Propagation in Eggs

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Influenza virus A/PR/8/1934 (H1N1) (PR8) were provided by Dr. Thomas Moran and Dr. Bruno Moltedo (Icahn School of Medicine at Mt. Sinai, New York)⁵⁵ (link) and were propagated in 10-day-old embryonated chicken eggs (Charles River Laboratories) for 48 hours at 37°C and tittered in MDCK cells.

Wu Q., Kumar N., Lafuse W.P., Ahumada O.S., Saljoughian N., Whetstone E., Zani A., Patton A.K., El Refaey M., Webb A., Pietrzak M., Yu L., KC M., Peeples M.E., Ganesan L.P., Yount J.S, & Rajaram M.V. (2022). Influenza A virus modulates ACE2 expression and SARS-CoV-2 infectivity in human cardiomyocytes. iScience, 25(12), 105701.

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Influenza infection in C57BL/6 and Ifnar1-/- mice

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C57BL/6 mice (6–8 weeks old) were obtained from Jackson Laboratory. Ifnar1^−/− mice [20 (link)] were a kind donation of Dr. Thomas Moran (Icahn School of Medicine at Mount Sinai) and were used with gender and age matched C57BL/6 mice (Jackson Laboratory bred in house). All experiments were performed in male and female mice. Influenza A/X-31 H3N2 (IAV X-31) and A/California/7/2009 H1N1 with D225G HA mutation (IAV-Cal/09-D225G) that allows the wild-type A/California/7/2009 virus to grow in eggs [21 (link)] were used as challenge strains. All strains of IAV were grown in 10 day-old embryonated chicken eggs (Charles River Laboratory) at 30,000 medium tissue culture infectious dose (TCID₅₀) at 37 °C. Allantoic fluid from infected eggs was collected 40 h later.

Fisher D.G., Coppock G.M, & López C.B. (2018). Virus-derived immunostimulatory RNA induces type I IFN-dependent antibodies and T-cell responses during vaccination. Vaccine, 36(28), 4039-4045.

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Propagation and Characterization of Avian Influenza Viruses

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The viruses A/duck/Wisconsin/480/1979 (H12N6; BEI Resources # NR-28616), A/redhead/Alberta/192/2002 (H3N6; BEI Resources # NR-45145), A/duck/England/1956 (H11N6; BEI Resources # NR-21660), A/gull/Maryland/704/1977 (H13N6; BEI Resources # NR-21663), A/black-legged kittiwake/Quebec/02838-1/2009 (H13N6; BEI Resources # NR-31217), A/ring-billed gull/Quebec/02434-1/2009 (H13N6; BEI Resources # NR-31218), A/mallard/Alberta/125/1999 (H11N6; BEI Resources # NR-45187), A/blue-winged teal/Illinois/10OS1546/2010 (H3N6; BEI Resources # NR-35981) and A/blue-winged teal/Wisconsin/402/1983 (H4N6; BEI Resources # NR-48971) were obtained from the Biodefense and Emerging Infections Research Resources Repository (BEI Resources). The viruses were propagated in 10 day old embryonated chicken eggs (Charles River Laboratories) at 37°C for 2 days. The viral titers were determined by conducting a standard plaque assay on Madin Darby canine kidney (MDCK) cells as described earlier (48 (link)). The viruses A/duck/Czechoslovakia/1956 (H4N6) and A/swine/Missouri/A01727926/2015 (H4N6) were rescued in the backbone of A/PR/8/34 as a 6:2 reassortant, containing the HA and NA segments of the original virus isolates (49 (link), 50 (link)).

Strohmeier S., Amanat F., Carreño J.M, & Krammer F. (2022). Monoclonal antibodies targeting the influenza virus N6 neuraminidase. Frontiers in Immunology, 13, 944907.

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Influenza B Virus Propagation and MDCK-Ln Cell Culture

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The following reagents used in this study were kindly provided by the International Reagent Resource (IRR): influenza B virus B/Phuket/3073/2013 (Phu/13) [FR-1364] and the Madin–Darby canine kidney cells, London line (MDCK-Ln) [FR-58]. The following virus was kindly provided by the Biodefense and Emerging Infectious Disease Repository (BEI): influenza B virus B/Florida/4/2006 (FL/4/06) [NR-9696]. Stock viruses obtained from IRR and BEI were used to infect the allantoic cavity 10 or 11 day-old embryonated chicken eggs (Charles River Laboratories, Wilmington, MA, USA) and incubated at 33–35 °C. Allantoic fluid was harvested 48–72 h after infection and virus was quantified through hemagglutinating units (HAU), aliquoted, and stored at −80 °C. Each virus was only subjected to one round of growth in embryonated eggs after arrival from IRR or BEI. MDCK-Ln cells were maintained in Dulbecco’s Modified Essential Medium (DMEM) (Cytiva) supplemented with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) (Cytiva) antibiotics and incubated at 37 °C in 5% CO₂.

Pekarek M.J., Petro-Turnquist E.M., Rubrum A., Webby R.J, & Weaver E.A. (2022). Expanding Mouse-Adapted Yamagata-like Influenza B Viruses in Eggs Enhances In Vivo Lethality in BALB/c Mice. Viruses, 14(6), 1299.

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