Htb 55

Manufactured by American Type Culture Collection

Sourced in United States

The HTB-55 is a cell culture incubator designed for maintaining and growing cell lines. It provides a controlled environment for temperature, humidity, and carbon dioxide levels to support optimal cell growth and proliferation.

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HTB-55TM (3 citations) ATCC HTB-55 (3 citations) Calu-3 (ATCC HTB-55), (2 citations) Calu-3 cells (HTB-55; (2 citations) Calu-3 (HTB-55; (2 citations)

53 protocols using htb 55

SARS-CoV-2 Variants Isolation and Characterization

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All cell lines were cultured in Dulbecco's Modified Eagle Medium containing 4.5 g/L or 1 g/L (Calu-3 cells) D-Glucose (DMEM; Thermo Fisher Scientific Inc., Pittsburgh, PA, USA). African green monkey epithelial kidney cells (VERO C1008, Vero 76, clone E6, ATCC CRL-1586) (Vero E6) and Vero E6 expressing Transmembrane Protease Serine 2 and Human Angiotensin-Converting Enzyme 2 (Vero E6-TMPRSS2-T2A-ACE2 cells; NR-54970) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco); human lung adenocarcinoma epithelial cells (Calu-3, ATCC HTB-55) were maintained in DMEM supplemented with 20% FBS; ACE2 transfected 293T (kindly given by PhD. Paul D. Bieniasz, The Rockfeller University, NY) were maintained in DMEM supplemented with 7.5% fetal bovine serum. Cells were cultured at 37 °C with 5% CO₂.
SARS-CoV-2 A2 (GISAID: 528539), Gamma, and Delta variants were isolated from symptomatic individuals cared at Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil) COVID-19 trial center. Isolation and sequencing of each variant were performed, as previously described (Voloch et al., 2021 (link)). The study was approved by the National Committee of Research Ethics (CAAE-30161620.0.1001.5257). Virus isolation, stock production and titration were performed on Vero E6 cells.

Bezerra B.B., Silva G.P., Coelho S.V., Correa I.A., Souza M.R., Macedo K.V., Matos B.M., Tanuri A., Matassoli F.L., Costa L.J., Hildreth J.E, & Arruda L.B. (2022). Hydroxypropyl-beta-cyclodextrin (HP-BCD) inhibits SARS-CoV-2 replication and virus-induced inflammatory cytokines. Antiviral Research, 205, 105373.

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Cell Lines Used for SARS-CoV-2 Research

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Grivet (Chlorocebus aethiops) kidney epithelial Vero (American Type Culture Collection [ATCC]; CCL-81) and Vero E6 (BEI Resources; NR596), Vero E6/TMPRSS2 (JCRB Cell Bank, Japan; JCRB1819), human colorectal adenocarcinoma Caco-2 (ATCC; HTB-37), HEK293T (ATCC; CRL-3216), human hepatocarcinoma HepG2 (ATCC; #HB-8065), human hepatocarcinoma Huh-7 (a gift from NIH/National Institute of Allergy and Infectious Diseases [NIAID]/Rocky Mountain Laboratories, Hamilton, Montana, USA), human lung adenocarcinoma Calu-3 (ATCC; HTB-55), and human lung carcinoma A549 cells (a gift from University of Rochester Medical Center) were maintained at 37°C and 5% CO₂ in DMEM (Life Technologies) containing 10% heat-inactivated FBS. Primary human hepatocytes from an anonymous 41-year-old woman with colorectal cancer metastasized to the liver were obtained from Liver Center Resources, Pittsburgh Liver Research Center, Pittsburgh, Pennsylvania, USA, and maintained at 37°C and 5% CO₂ in Eagle’s modified essential medium (Gibco).

Bennett R.P., Postnikova E.N., Eaton B.P., Cai Y., Yu S., Smith C.O., Liang J., Zhou H., Kocher G.A., Murphy M.J., Smith H.C, & Kuhn J.H. (2022). Sangivamycin is highly effective against SARS-CoV-2 in vitro and has favorable drug properties. JCI Insight, 7(1), e153165.

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Cultivation and Titration of SARS-CoV-2 and Common Coronaviruses

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Human lung fibroblasts MRC5 (ATCC CCL-171), the human colorectal carcinoma HCT-8 (ATCC CCL-244), the human lung adenocarcinoma Calu-3 (ATCC HTB-55), and the African green monkey kidney Vero E6 (ATCC CRL-1586) cell lines were purchased from the American Type Culture Collection (ATCC), and maintained in Dulbecco’s Modified Eagle Medium (DMEM; Euroclone) supplemented with 10% fetal bovine serum (FBS, Euroclone), 2 mM glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate (P/S, both from Euroclone).
hCoV-229E (ATCC VR-740) and hCoV-OC43 (ATCC VR-1558) were purchased from ATCC, and propagated and titrated in MRC5 and HCT-8 cells, respectively. SARS-CoV-2 (2019-nCoV/Italy INMI1) was obtained from EVAg, and propagated and titrated in Vero E6 cells. SARS-CoV-2/01/human/2020/SWE was isolated on Vero E6 cells from a nasopharyngeal sample, cultivated and titrated as previously described [18 (link)]. All work with SARS-CoV-2 was performed in Biosafety laboratory level 3 (BSL3) facilities either at the University of Padua, Italy, or at the Public Health Agency of Sweden, Sweden.

Calistri A., Luganini A., Mognetti B., Elder E., Sibille G., Conciatori V., Del Vecchio C., Sainas S., Boschi D., Montserrat N., Mirazimi A., Lolli M.L., Gribaudo G, & Parolin C. (2021). The New Generation hDHODH Inhibitor MEDS433 Hinders the In Vitro Replication of SARS-CoV-2 and Other Human Coronaviruses. Microorganisms, 9(8), 1731.

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Comparative Study of SARS-CoV-2 and HCoV-OC43 in Cell Lines

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The African monkey kidney Vero E6 (ATCC® CRL-1586™) and Vero (ATCC® CCL-81™) cells, human lung epithelial cells Calu-3 (ATCC® HTB-55™), human epithelial colorectal adenocarcinoma cells Caco-2 (ATCC® HTB-37™), human kidney epithelial cells 293T/17 (ATCC® CRL-11268™), baby golden hamster kidney fibroblasts BHK-21 (ATCC® CCL-10™) and mouse embryonic fibroblasts 3T3 (ATCC® CRL-1658™) were purchased from ATCC (Manassas, VA). Human hepatocellular carcinoma cells Huh-7 (JCRB0403) was purchased from JCRB Cell Bank (Japan). Primary human airway epithelium cells (HAE) from healthy donors were obtained from Lonza Biosciences (CC-2540s) and cultured in air-liquid interface by Candela Manfredi in Dr. Eric Sorscher's group at Emory University. SARS-CoV-2 was provided by BEI Resources (NR-52281: USA-WA1/2020) and HCoV-OC43 was obtained from ATCC (Manassas, VA).

Tao S., Zandi K., Bassit L., Ong Y.T., Verma K., Liu P., Downs-Bowen J.A., McBrayer T., LeCher J.C., Kohler J.J., Tedbury P.R., Kim B., Amblard F., Sarafianos S.G, & Schinazi R.F. (2021). Comparison of anti-SARS-CoV-2 activity and intracellular metabolism of remdesivir and its parent nucleoside. Current Research in Pharmacology and Drug Discovery, 2, 100045.

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SARS-CoV-2 Infection of Human Cell Lines

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The human B lymphocytes (GM12878) were purchased from the NGIMS Human Genetics Cell Repository, Cornell Institute of Medical Research (Camden, NJ). The B cells were maintained as suspension cultures at 37°C in Advanced RPMI-1640 medium supplemented with 15% of fetal bovine serum (FBS), L-glutamine, (Gibco™, Carlsbad, CA), and 5% CO₂. The medium was refreshed every two to three days to maintain a cell density of 10⁶ ml⁻¹.
A549 cells were obtained from ATCC (CRL-1586 and CCL-185, respectively) and were grown in Dulbecco’s modified Eagle medium (DMEM) containing 2 mM L-glutamine, non-essential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal calf serum (DMEM complete). Calu-3 cells (ATCC, HTB-55) were a kind gift from Dr. Manfred Frey, Mannheim, and were cultured in DMEM complete with 20% fetal calf serum. HEK293-ACE2 cells were a kind gift from Prof. Stephan Becker at University of Marburg. A549 cells stably expressing ACE2 (A549-ACE2) were generated as described recently (Klein et al., 2020 (link)). SARS-CoV-2 isolate Bavpat1/2020 was kindly provided by Prof. Christian Drosten (Charité Berlin, Germany) through the European Virology Archive (Ref-SKU: 026V-03883) at passage 2. Working virus stocks were produced as described previously (Klein et al., 2020 (link)) by passaging the virus two times in VeroE6 cells.

Loconte V., Chen J.H., Cortese M., Ekman A., Le Gros M.A., Larabell C., Bartenschlager R, & Weinhardt V. (2021). Using soft X-ray tomography for rapid whole-cell quantitative imaging of SARS-CoV-2-infected cells. Cell Reports Methods, 1(7), 100117.

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Cytotoxic Effects of Asbestos on Lung Cells

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Human lung cancer cell lines (NCI-H358, ATCC ® CRL-5807, lung bronchioloalveolar carcinoma; Calu-3, ATCC ® HTB-55, lung adenocarcinoma; A549, ATCC ® CCL-185, lung adenocarcinoma) and IMR-90 human lung fibroblast cell lines (ATCC ® CCL-186) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). IMR-90 and Calu-3 cells were cultured in Eagle’s minimal essential medium (EMEM), NCI-H358 cells in Roswell Park Memorial Institute (RPMI)-1640 medium, and A549 cells in F-12K medium supplemented with 10% fetal bovine serum (FBS, WelGENE Inc., Deagu, South Korea) and 100 U/mL penicillin-streptomycin (WelGENE Inc.) in a humidified incubator set at 37°C with 5% CO₂. Subsequently, NCI-H358 and A549 cells were cultured in EMEM to match the medium used for IMR-90 cells. We performed cytotoxicity and cell viability assays as well as real-time cell monitoring for 120 hours to observed the effects of exposure to 50 mg/L chrysotile, 50 mg/L crocidolite, and 50 mg/L amosite on IMR-90 making IMR-90 being cytostatic, and decided to use 50mg/L as our experimental concentration for all types of asbestos [17 ]. The study was approved by the institutional review board of Yeouido St. Mary’s Hospital (Korea; approval number SC19ZNSI0015) and was in accordance with the relevant legislation.

Yu S., Choi H.H., Kim I.W, & Kim T.J. (2019). Conditioned medium from asbestos-exposed fibroblasts affects proliferation and invasion of lung cancer cell lines. PLoS ONE, 14(9), e0222160.

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Cell Culture Protocols for Multiple Cell Lines

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Vero E6 cells, Vero CCL-81 cells (ATCC^® CCL-81™), and human lung carcinoma cells (A549; ATCC^® CRL-185™) were cultured in Dulbecco’s minimal essential medium (DMEM; Gibco, Grand Island, NE, USA) supplemented with HEPES (Sigma Aldrich, St. Louis, MO, USA), 10% heat-inactivated fetal bovine serum (FBS; Gibco, Paisley, UK), and penicillin-streptomycin (10,000 U/mL) (Gibco, Grand Island, NE, USA). Human fibroblast cells (MRC-5; ATCC^® CRL-171™) and human airway epithelial cells (Calu-3; ATCC^® HTB-55™) were maintained in Eagle’s minimum essential medium (EMEM; ATCC, Manassas, VA, USA) supplemented with 10% heat-inactivated FBS and penicillin-streptomycin. The cells were passaged every 2 to 3 days by dissociating them with TrypLE™ Express Enzyme (Gibco, Grand Island, NE, USA). All cells were grown in a humidified 37 °C incubator with 5% CO₂.

Rosli S.N., Dimeng S.R., Shamsuddin F., Mohd Ali M.R., Muhamad Hendri N.A., Suppiah J., Mohd Zain R., Thayan R, & Ahmad N. (2023). Vero CCL-81 and Calu-3 Cell Lines as Alternative Hosts for Isolation and Propagation of SARS-CoV-2 Isolated in Malaysia. Biomedicines, 11(6), 1658.

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Investigating AMPK Signaling in COVID-19

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Calu3 (HTB-55™; ATCC) and Vero (CCL-81™; ATCC) cells were grown in DMEM supplemented with 10% FBS and 1 × Pen Strep, and Caco2 (HTB-37™; ATCC) cells were grown in DMEM supplemented with 20% FBS and 1 × Pen-Strep, at 37 °C and 5% CO₂. Rabbit monoclonal antibodies against AMPK and phospho-AMPK (T172) were procured from CST. Mouse monoclonal GAPDH, β-tubulin antibodies, and rabbit monoclonal Nucleocapsid antibodies were purchased from ThermoFisher Scientific. HRP-conjugated secondary antibodies were purchased from Jackson ImmunoResearch. Metformin, AICAR, and Compound C were procured from Merck Millipore.

Parthasarathy H., Tandel D., Siddiqui A.H, & Harshan K.H. (2022). Metformin suppresses SARS-CoV-2 in cell culture. Virus Research, 323, 199010.

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Influenza Virus Culture and Titration in Cell Lines

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The Madin–Darby canine kidney (MDCK) (ATCC CCL-34), the human adenocarcinoma alveolar basal epithelial A549 (ATCC CCL-185), and the human lung adenocarcinoma Calu-3 (ATCC HTB-55) cell lines were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle medium (DMEM; Euroclone (Milan, Italy)) supplemented with 10% fetal bovine serum (FBS, Euroclone), 2 mM glutamine, 1 mM sodium pyruvate, and 100 U/mL penicillin and 100 μg/mL streptomycin sulfate (P/S, both from Euroclone). All IV infections were performed in the presence of 2 μg/mL of trypsin TPCK treated by bovine pancreas (Sigma-Aldrich) and 0.14% of bovine serum albumin (Sigma-Aldrich).
The Influenza A virus strain A/Puerto Rico/8/34 (PR8) H1N1 (IAV) and influenza B virus strain B/Lee/40 (IBV) were a generous gift from Arianna Loregian (University of Padua, Padua, Italy). IAV and IBV were propagated and titrated by the plaque assay on MDCK cells, as previously described [32 (link),33 (link)].

Sibille G., Luganini A., Sainas S., Boschi D., Lolli M.L, & Gribaudo G. (2022). The Novel hDHODH Inhibitor MEDS433 Prevents Influenza Virus Replication by Blocking Pyrimidine Biosynthesis. Viruses, 14(10), 2281.

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Cell Lines for SARS-CoV-2 Research

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African Green monkey kidney cells VeroE6 expressing ACE-2 (VeroE6-ACE2) alone and with TMPRSS2 (VeroE6-ACE2-TMPRSS2), BHK-21, and A549NPro expressing ACE-2 (A549NPro-ACE2) were grown in DMEM-Glutamax supplemented with 10% fetal calf serum (FCS; Gibco) and non-essential amino acids (NEAA; Gibco). VeroE6 were a kind gift from Professor M. Bouloy (Institut Pasteur, France), BHK were sourced from the ATCC (ATCC CCL-10) and, VeroE6-ACE2, and VeroE6-ACE2-TMPRSS2 have been described elsewhere [66 (link)] A549NPro cells (a kind gift from Prof. R. Randall, University of St. Andrews, UK) were transduced to stably express ACE-2 (A549NPro-ACE2) as described [66 (link)]. Calu-3, a human lung epithelial cell line was grown in DMEM Glutamax, supplemented with 20%(v/v) FCS and NEAA was sourced from the ATCC (ATCC HTB-55). All cells were maintained at 37°C in 5%(v/v) CO₂, humidified incubator.

Szemiel A.M., Merits A., Orton R.J., MacLean O.A., Pinto R.M., Wickenhagen A., Lieber G., Turnbull M.L., Wang S., Furnon W., Suarez N.M., Mair D., da Silva Filipe A., Willett B.J., Wilson S.J., Patel A.H., Thomson E.C., Palmarini M., Kohl A, & Stewart M.E. (2021). In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2. PLoS Pathogens, 17(9), e1009929.

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