Us mo 14 18947

Manufactured by American Type Culture Collection

US/MO/14-18947 is a laboratory equipment product. It is designed for use in scientific research and testing applications. The core function of this product is to facilitate specific tasks or procedures within a laboratory setting. However, a detailed description of its features and intended use cannot be provided while maintaining an unbiased and purely factual approach.

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6 protocols using us mo 14 18947

Culturing and Amplification of Enteroviruses

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Rhabdomyosarcoma (RD, ATCC, CCL-136), A172 (ATCC, CRL-1620) and SH-SY5Y (ATCC, CRL-2266) were maintained in a 37 °C incubator in a 5% CO₂ atmosphere. RD cell was cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S). SH-SY5Y were cultured in 10% FBS and 1% P/S with 50% DMEM and 50% F-12 medium. All of the following EV-D68 strains used in this study were purchased from ATCC: US/KY/14-18953 (ATCC, NE-49132), US/MO/14-18947 (ATCC, NR-49129), US/MO/14-18949 (ATCC, NR-49130), US/IL/14-18952 (ATCC, NR-49131), US/IL/14-18956 (ATCC, NR-49133). All of the following EV-A71 strains used in this study were purchased from ATCC or BEI Resources: Tainan/4643/1998 (BEI Resources, NR-471), Enterovirus 71, MP4 (BEI Resources, NR-472). Enteroviruses A71 US/CT/2016- 19519 was obtained from Dr. William Nix at the Centers for Disease Control and Prevention under a material transfer agreement. All viruses were amplified in RD cells prior to infection assays.

Hu Y., Kitamura N., Musharrafieh R, & Wang J. (2021). Discovery of potent and broad-spectrum pyrazolopyridine-containing antivirals against enterovirus D68, A71, and coxsackievirus B3 by targeting the viral 2C protein. Journal of medicinal chemistry, 64(12), 8755-8774.

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Rhabdomyosarcoma and EV-D68 Virus Culture

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Rhabdomyosarcoma (RD, ATCC, CCL-136), A172 (ATCC, CRL-1620), A549 (ATCC,
CCL-185), HeLa (ATCC, CCL-2), and SH-SY5Y (ATCC, CRL-2266) were maintained in a
37 °C in a 5% CO₂ atmosphere. RD and A172 were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine
serum (FBS) and 1% penicillin-streptomycin (P/S). SH-SY5Y were cultured in 10%
FBS and 1% P/S with 50% DMEM and 50% F-12 medium. All of the following EV-D68
strains used in this study were purchased from ATCC: US/KY/14-18953 (ATCC,
NE-49132), US/MO/14-18947 (ATCC, NR-49129), US/MO/14-18949 (ATCC, NR-49130),
US/IL/14-18952 (ATCC, NR-49131), US/IL/14-18956 (ATCC, NR-49133).

Musharrafieh R., Zhang J., Tuohy P., Kitamura N., Bellampalli S.S., Hu Y., Khanna R, & Wang J. (2019). Discovery of quinoline analogs as potent antivirals against enterovirus D68 (EV-D68). Journal of medicinal chemistry, 62(8), 4074-4090.

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Propagation and Maintenance of EV-D68 and EV-A71 Viruses

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The Fermon (ATCC, VR-1826), US/KY/14-18953 (ATCC, VR-1825D), and US/MO/14-18947 (ATCC, VR-1823D) strains of EV-D68; and the Changchun077 strain of EV-A71 have been reported previously (Wang et al., 2012 (link)). Viruses were propagated in human rhabdomyosarcoma RD cells (No CCL-136), and the supernatants were harvested and stored at −80°C. Human embryonic kidney cells (HEK 293T cells) (No CRL-11268) and RD cells were purchased from the ATCC (Manassas, VA, USA) and used according to a previous study (Wang et al., 2015 (link)). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO BRL, Grand Island, NY, USA).

Wang Z.Y., Zhong T., Wang Y., Song F.M., Yu X.F., Xing L.P., Zhang W.Y., Yu J.H., Hua S.C, & Yu X.F. (2017). Human Enterovirus 68 Interferes with the Host Cell Cycle to Facilitate Viral Production. Frontiers in Cellular and Infection Microbiology, 7, 29.

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Enterovirus Isolation and Purification Protocol

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EV-D68 prototype Fermon (2014) isolated US/MO/14-18947 (MO) and US/KY/14-18953 (KY) were purchased from ATCC. Professor Cheng Tong kindly provided the EV-A71 prototype Anhui2007, and prototype CC063 was isolated in our laboratory. Viruses propagated in RD cells. The supernatants of enterovirus-infected cells were harvested, filtered through a 0.22 mm filter, and centrifuged through a 20% sucrose cushion in an SW28 rotor at 28,000 rpm for 1.5 h. Purified pellets were stored at −80°C.

Li Y., Shen S., Guo H., Zhang Z., Zhang L., Yang Q., Gao Y., Niu J, & Wei W. (2021). Enterovirus Infection Restricts Long Interspersed Element 1 Retrotransposition. Frontiers in Microbiology, 12, 706241.

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Enterovirus D68 Strain Propagation

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Three strains of enterovirus D68 were obtained: US/MO/14-18947 (purchased from BEI Resources, NR-49129, lot no. 63205984), US/IL/14-18952 (purchased from BEI Resources, NR-49131, lot no. 63205985), and EV-D68 Fermon strain (generous gift from Charles Y. Chiu at the University of California, San Francisco, CA). Enterovirus D68 strains were grown in human muscle rhabdomyosarcoma (RD) cells (purchased from ATCC CCL-136) for all experiments except those shown in Fig. 5, for which US/MO/14-18947 was grown in SH-SY5Y neuroblastoma cells (purchased from ATCC CRL-2266). Virus was added at a multiplicity of infection (MOI) of 100 to RD cells in a T182 flask for 60 min, followed by a phosphate-buffered saline (PBS) rinse and incubation in 10 mL medium (95% high-glucose Dulbecco’s modified eagle medium [DMEM] + 5% fetal bovine serum [FBS]) for 3 days at 33°C. All strains killed 99% of RD cells in the flask after 3 days.

Rudy M.J., Coughlan C., Hixon A.M., Clarke P, & Tyler K.L. (2022). Density Analysis of Enterovirus D68 Shows Viral Particles Can Associate with Exosomes. Microbiology Spectrum, 10(1), e02452-21.

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Culturing RD and Vero Cells for Enterovirus Assays

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RD and Vero Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) as described previously^{56 (link),57 (link)}. CV-A10 strains S0148b, S0273b, and Kowalik have been described in a previous study^{24 (link)}. EV-A71/G082 and CV-A16/SZ05 have been described previously^{54 (link)}. EV-D68 strains US/MO/14-18947 (ATCC Number: VR-1823), US/KY/14-18953 (VR-1825), and Fermon (VR-1826) were obtained from ATCC. CV-B3 strain Nancy and PV-1 strain Sabin have been described in a previous study^{58 (link)}. All virus stocks were made in RD cells. Virus titers were determined according to the Reed–Muench method⁵⁹ and expressed as 50% tissue culture infectious dose (TCID50).

Chen J., Ye X., Zhang X.Y., Zhu Z., Zhang X., Xu Z., Ding Z., Zou G., Liu Q., Kong L., Jiang W., Zhu W., Cong Y, & Huang Z. (2019). Coxsackievirus A10 atomic structure facilitating the discovery of a broad-spectrum inhibitor against human enteroviruses. Cell Discovery, 5, 4.

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