Specific pathogen free embryonated chicken eggs

Manufactured by Charles River Laboratories

Sourced in Holy See (Vatican City State), United States

Specific pathogen-free embryonated chicken eggs are a type of laboratory equipment used in various research and testing applications. They provide a controlled and standardized substrate for the cultivation and study of microorganisms, viruses, and other biological agents. These eggs are derived from chickens that have been screened and confirmed to be free of specific pathogens, ensuring a reliable and consistent environment for experimentation.

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Lab products found in correlation

Miseq ngs platform, by Illumina (1 mentions) Avertin, by Merck Group (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions) Thp 1, by American Type Culture Collection (1 mentions) Dulbecco modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions) Sw28 rotor, by Beckman Coulter (1 mentions)

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Embryonated chicken eggs (37 citations) Specific‐pathogen free eggs (7 citations) Chicken eggs (6 citations) Specific-pathogen-free chicken eggs (2 citations)

9 protocols using specific pathogen free embryonated chicken eggs

Propagation and Characterization of H5N1 Virus

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Highly pathogenic avian influenza A/Vietnam/1203/2004 (H5N1) virus was kindly provided by Dr. S. Mark Tompkins at the Department of Infectious Diseases, University of Georgia. Virus propagation was carried out under BSL3 conditions at the University of Georgia. Virus stock was amplified in 10-11 day-old embryonated specific pathogen free chicken eggs (Charles River Laboratories) for 24 h as previously described (33 (link)). Allantoic fluid from inoculated eggs was clarified by centrifugation and aliquots stored at −80°C until transferred to the University of Pittsburgh Regional Biocontainment Laboratory BSL-3 facility. Virus titer was determined in Madin-Darby canine kidney cells (MDCK) by plaque assay and expressed as pfu/ml. Whole genome sequencing of virus seed and propagated stocks was performed using the Illumina MiSeq NGS platform. A comparison of the consensus sequence to publicly available A/Vietnam/1203/2004 (H5N1) sequences on the Influenza Research Database (http://www.fludb.org/) showed no amino acid changes in the virus seed and propagated stocks used in this experiment. Virus was diluted in media containing bovine serum albumin, HEPES buffer, penicillin/streptomycin and trypsin immediately prior to aerosol exposure.

Wonderlich E.R., Swan Z.D., Bissel S.J., Hartman A.L., Carney J.P., O’Malley K.J., Obadan A.O., Santos J., Walker R., Sturgeon T.J., Frye LJ J.r., Maiello P., Scanga C.A., Bowling J.D., Bouwer A.L., Duangkhae P.A., Wiley C.A., Flynn J.L., Wang J., Cole K.S., Perez D.R., Reed D.S, & Barratt-Boyes S.M. (2017). Widespread virus replication in alveoli drives acute respiratory distress syndrome in aerosolized H5N1 influenza infection of macaques. Journal of immunology (Baltimore, Md. : 1950), 198(4), 1616-1626.

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Intranasal IAV Infection in Chemical-Exposed Mice

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Following 8 weeks of exposure to the chemical mixture or water containing the vehicle control, mice were anesthetized by intraperitoneal (IP) injection of avertin (2% 2,2,2-tribromoethanol; Sigma). Anesthetized mice were infected intranasally (IN) with 120 hemagglutinating units (HAU) of IAV (HKx31; H3N2). The virus stock was initially obtained from M. Coppola (Argonex, Charlottesville, VA), propagated in embryonated specific-pathogen free chicken eggs (Charles River, Wilmington. MA), and titered using a hemagglutination assay (Barrett and Ingles 1985 ). For infection, IAV was diluted in sterile endotoxin-tested phosphate-buffered saline (PBS, pH 7.4). Mice were maintained on drinking water containing the chemical mixture or vehicle control following infection. Morbidity and mortality were monitored daily, starting on the day of infection. Using flow cytometry, T-cells in the mediastinal lymph nodes (MLN) were examined on the peak day of host response to IAV infection, which is Day 8 in male and Day 9 in female mice (Boule et al. 2014 (link); Lawrence et al. 2000 , 2006 (link)). All work with infectious agents was conducted with prior approval of the Institutional Biosafety Committee of the University of Rochester, following guidelines of the NIH/CDC.

O’Dell C.T., Boule L.A., Robert J., Georas S.N., Eliseeva S, & Lawrence B.P. (2021). Exposure to a Mixture of 23 Chemicals Associated with Unconventional Oil and Gas Operations Alters Immune Response to Challenge in Adult Mice. Journal of immunotoxicology, 18(1), 105-117.

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Plaque Assays Using MDCK Cells

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Madin-Darby Canine Kidney (MDCK) cells used for plaque assays were maintained in Minimum Essential Media supplemented with 10% fetal bovine serum (FBS, Hyclone) and 100 U/mL penicillin plus 100 μg/mL streptomycin (1x P/S). Virus stocks were grown in 8-day-old specific pathogen-free embryonated chicken eggs (Charles River Laboratories) at 37°C for 2 days or at 33°C for 3 days, and were then chilled down to 4°C overnight. Allantoic fluid was harvested, centrifuged at 290 × g for 5 min, and titrated by plaque assays as previous study described (19 (link)). Plaques were visualized by immunostaining or crystal violet staining. Recombinant proteins utilized in this study consist of chimeric H6/1 [containing H6 head domain from A/mallard/Sweden/81/02 combined with an H1 stalk domain of A/California/04/09 (Cal/09)], H1 (Cal/09), H2 (A/mallard/Netherlands/5/99), H3 (A/Hong Kong/4801/14), H6 (A/mallard/Sweden/81/02), H18 (A/bat/Peru/33/10), and N1 (Cal/09). Recombinant proteins were expressed in High Five cells grown in serum free SFX medium (HyClone) using the baculovirus expression system (49 (link)).

Liu W.C., Nachbagauer R., Stadlbauer D., Solórzano A., Berlanda-Scorza F., García-Sastre A., Palese P., Krammer F, & Albrecht R.A. (2019). Sequential Immunization With Live-Attenuated Chimeric Hemagglutinin-Based Vaccines Confers Heterosubtypic Immunity Against Influenza A Viruses in a Preclinical Ferret Model. Frontiers in Immunology, 10, 756.

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Cell Lines and Virus Strains Used

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Human embryonic kidney 293 T cells (American Type Culture Collection; ATCC) (cat. no. CRL-1573), Madin-Darby Canine Kidney (MDCK) (ATCC; cat. no. CCL-34) cells, and adenocarcinomic human alveolar basal epithelial cells (A549) (ATCC; cat. no. CCL-185) were grown in Dulbecco modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) (HyClone) and 100 U/mL of penicillin and streptomycin (Gibco). THP-1 (ATCC) cells (ATCC; cat. no. TIB-202) were maintained in RPMI-1640 (Gibco) supplemented with 10% FBS and 100 U/mL of penicillin and streptomycin (Gibco). A/Shanghai/1/13, X31, and X79 are reassortant viruses that have six internal proteins of A/Puerto Rico/8/34 expressing the HA and NA of the indicated viruses: X31 (HA and NA of HK/68, H3N2), X79 (HA and NA of A/Philippines/2/82, H3N2), and H7N9 (HA and NA of A/Shanghai/1/13) that have been passaged and grown at the Icahn School of Medicine at Mount Sinai. Other viruses utilized in the paper are: A/Victoria/361/11 (Vic/11) (H3N2), A/Perth/16/09 (H3N2), and A/Netherlands/602/2009 (NL09) (H1N1) (these viruses were obtained from the Centers of Disease Control and Prevention). All the viruses above were grown in 10-day-old specific-pathogen-free embryonated chicken eggs (Charles River Laboratories, Inc.).

He W., Chen C.J., Mullarkey C.E., Hamilton J.R., Wong C.K., Leon P.E., Uccellini M.B., Chromikova V., Henry C., Hoffman K.W., Lim J.K., Wilson P.C., Miller M.S., Krammer F., Palese P, & Tan G.S. (2017). Alveolar macrophages are critical for broadly-reactive antibody-mediated protection against influenza A virus in mice. Nature Communications, 8, 846.

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Virus Purification from Embryonated Eggs

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Viruses were grown in 10-day-old specific-pathogen-free embryonated chicken eggs (Charles River) for 48 h at 37°C. Allantoic fluid was harvested and subjected to low-speed centrifugation (relative centrifugal force [RCF] of 3,000 for 30 min at 4°C) to remove cellular debris. Viruses were pelleted through a 30% sucrose cushion (30% sucrose in NTE buffer [100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA] [pH 7.4]) by ultracentrifugation (25,000 rpm for 2 h at 4°C using a Beckman SW28 rotor). After aspiration of the supernatant, virus pellets were resuspended in phosphate-buffered saline (PBS). When necessary, virus preparations were inactivated with 0.03% formalin for 48 h at 4°C.

Tran E.E., Podolsky K.A., Bartesaghi A., Kuybeda O., Grandinetti G., Wohlbold T.J., Tan G.S., Nachbagauer R., Palese P., Krammer F, & Subramaniam S. (2016). Cryo-electron Microscopy Structures of Chimeric Hemagglutinin Displayed on a Universal Influenza Vaccine Candidate. mBio, 7(2), e00257-16.

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Influenza Virus Protein Production and Assays

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The methods for Madin-Darby Canine Kidney (MDCK) cell culture and plaque assays were described previously [31 (link)]. Virus stocks were prepared in 8-day-old specific pathogen-free embryonated chicken eggs (Charles River Laboratories), as previously described [31 (link)]. Influenza virus plaques were visualized by immunostaining or crystal violet staining [31 (link)]. The cH6/1 (containing H6 head domain from A/mallard/Sweden/81/02 combined with an H1 stalk domain of A/California/04/09 (Cal/09)), H1 (Cal/09), H2 (A/mallard/Netherlands/5/99), H6 (A/mallard/Sweden/81/02), and H18 (A/bat/Peru/33/10) recombinant proteins were produced by a baculovirus expression system as described previously [37 (link),38 (link)].

Liu W.C., Nachbagauer R., Stadlbauer D., Strohmeier S., Solórzano A., Berlanda-Scorza F., Innis B.L., García-Sastre A., Palese P., Krammer F, & Albrecht R.A. (2021). Chimeric Hemagglutinin-Based Live-Attenuated Vaccines Confer Durable Protective Immunity against Influenza A Viruses in a Preclinical Ferret Model. Vaccines, 9(1), 40.

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Influenza Virus Propagation in Eggs

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H1N1 strains of influenza virus A/Puerto Rico/8/34 (PR/8) and A/WSN/1933 (WSN), and clinical isolates H1N1 A/California/07/2009 (pdm/CA/09) and H1N1 A/Oklahoma/3052/2009 (A/OK/09) were propagated in the allantoic cavities of specific-pathogen-free embryonated chicken eggs (Charles River Laboratories, Wilmington, MA, USA) at 35 °C for 10 days. The allantoic fluids were collected, centrifuged at 2000× g for 10 min, and stored at −80 °C. Virus titer was determined using a tissue culture infective dose (TCID₅₀) assay [27 (link)].

Zhu Z., Yang X., Huang C, & Liu L. (2023). The Interferon-Induced Protein with Tetratricopeptide Repeats Repress Influenza Virus Infection by Inhibiting Viral RNA Synthesis. Viruses, 15(7), 1412.

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Propagation and Inactivation of Influenza Viruses

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A/Victoria/361/2011 (Vic/11; H3N2) (BEI Resources) and A/California/07/2009 (Cal/09: H1N1) (BEI Resources) were propagated in 10-day-old specific pathogen-free embryonated chicken eggs (Charles River Laboratories Wilmington, MA) at 35˚C and with 55-65% humidity. Allantoic fluid was harvested at 48 HPI followed by overnight incubation at 4˚C. Each of the Vic/11 and Cal/09 batches was grown from a seed stock and multiple batches were tested. Infectious influenza titer was determined by a standard plaque assay on MDCK cells in the presence of 2 µg/mL L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin. IAV inactivation was performed by delivering 2400 µJ/cm² of UV light (254 nm) using a Strata linker 1800 (Stratagene, La Jolla, CA.). Virus inactivation was verified by plaque assay or by intracellular influenza nucleoprotein antibody staining followed by analysis by flow cytometry.

Harvey A.G., Graves A.M., Uppalapati C.K., Matthews S.M., Rosenberg S., Parent E.G., Fagerlie M.H., Guinan J., Lopez B.S, & Kronstad L.M. (2022). Dendritic cell-natural killer cell cross-talk modulates T cell activation in response to influenza A viral infection. Frontiers in Immunology, 13, 1006998.

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Propagation and Inactivation of Influenza Viruses

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A/Victoria/361/2011(Vic/11; H3N2), A/California/07/2009 (Cal/09: H1N1), A/Bayern/7/1995 (Bay/95; H1N1) (Influenza Reagent Resource), A/Beijing/32/1992 (Beij/92; H3N2), A/Hong Kong/1/68 (HK/68; H3N2) and A/Melbourne/1/1946 (Mel/46; H1N1) were propagated in 10-day-old specific pathogen-free embryonated chicken eggs (Charles River Laboratories International, Inc.) at 35°C and 55–65% humidity. Allantoic fluid was harvested 48 hours post-infection. Each Vic/11 and Cal/09 batch was grown from a seed stock and multiple batches were tested. A/Puerto Rico/8/34 (H1N1) was grown in Madin-Darby canine kidney (MDCK) cells. Infectious influenza titer was determined by standard plaque assay on MDCK cells in the presence of 2 µg/mL L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin. Influenza A virus inactivation was performed by delivering 1800 mJ of UV light (254 nm) using a Stratalinker 1800 (Stratagene, La Jolla, Calif.); inactivation was verified by MDCK plaque assay.

Kronstad L.M., Seiler C., Vergara R., Holmes S.P, & Blish C.A. (2018). Differential induction of IFN-α and modulation of CD112 and CD54 expression governs the magnitude of NK cell IFN-γ response to influenza A viruses. Journal of immunology (Baltimore, Md. : 1950), 201(7), 2117-2131.

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