Ccf sttg1

Manufactured by American Type Culture Collection

Sourced in United States

The CCF-STTG1 is a cell culture flask designed for the growth and maintenance of cell lines. It provides a controlled environment for the cultivation of cells, enabling researchers to study their behavior and characteristics. The core function of the CCF-STTG1 is to serve as a reliable and standardized cell culture vessel.

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12 protocols using ccf sttg1

Cultivation and Differentiation of Human Cell Lines

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The human neuroblastoma cell line SHSY5Y (CRL-2266), the human astrocytoma cell line CCF-STTG1 (CRL-1718), human monocytic leukemia cell line THP-1 (TIB-202), and the human lung carcinoma cell line A549 (CCL-185) were obtained from the American Type Culture Collection (ATCC). SHSY5Y cells were cultured in a 1:1 mixture of EMEM (ATCC 30–2003) and F-12K (ATCC 30–2004) supplemented with 10% fetal bovine serum (FBS) (ATCC 30–2020). CCF-STTG1 and THP-1 cells were cultured in RPMI-1640 (ATCC 30–2001) supplemented with 10% FBS. A549 cells were cultured in F-12K (ATCC 30–2004) supplemented with 10% FBS. Cells were maintained in culture following the manufacturer’s recommendations. HIV-1 infected U937 Cells (U1; NIH AIDS Reagent Program) were cultured in RPMI-1640 supplemented with 10% exosome-depleted FBS. To generate MDMs, THP-1 cells and U1 cells were treated with phorbol 12-myristate 13-acetate (PMA; 100 nM) and incubated for four to five days. U1-MDMs then received two treatments of cART (emtricitabine, tenofovir disoproxil fumarate, ritonavir, darunavir; 2.5 µM each) over a five day incubation period.

Branscome H., Khatkar P., Al Sharif S., Yin D., Jacob S., Cowen M., Kim Y., Erickson J., Brantner C.A., El-Hage N., Liotta L.A, & Kashanchi F. (2022). Retroviral infection of human neurospheres and use of stem Cell EVs to repair cellular damage. Scientific Reports, 12, 2019.

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Generating Infectious HEV Strains for Research

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A subclone of the human hepatoma Huh7 cell line, Huh7-S10-3, a gift from Suzanne U. Emerson, NIH, Bethesda, MD, was used for generating infectious HEV RNA for transfection in this study. Another human hepatoma cell line, HepG2, was purchased from ATCC and used to propagate HEV. Both cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) at 37 °C with 5% CO₂. A human astrocyte-derived cell line (CCF-STTG1) was purchased from ATCC and cultured in DMEM with 10% FBS. A human brain microvascular endothelial cell line (hCMEC/D3), which is derived from human temporal lobe microvessel endothelial cells (Millipore, SCC066), was cultured in a collagen-coated flask or plate with hCMEC/D3-specific EndoGRO-MV complete media (Millipore, SCME004) containing human fibroblast growth factor (65 (link)). Genotype 3 HEV strain Kernow-C1/P6 (designated as P6) was rescued by transfection of Huh7-S10-3 cells with in vitro-transcribed RNAs from an infectious complementary DNA (cDNA) clone of the HEV P6 strain (66 (link)). The virus stock of another human-origin genotype 3 HEV (strain US2) was prepared in 10% PBS suspension of feces from experimentally infected pigs (67 (link)).

Tian D., Li W., Heffron C.L., Wang B., Mahsoub H.M., Sooryanarain H., Hassebroek A.M., Clark-Deener S., LeRoith T, & Meng X.J. (2022). Hepatitis E virus infects brain microvascular endothelial cells, crosses the blood–brain barrier, and invades the central nervous system. Proceedings of the National Academy of Sciences of the United States of America, 119(24), e2201862119.

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Paraffin Embedded Cell Line CPAs

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Cell lines PC3 (ATCC: CRL-1435), A549 (ATCC: CCL-185), HeLa (ATCC: CCL-2), Pfeiffer (ATCC: CRL-2632), OAW28 (Sigma: 85101601), CCFSTTG1 (ATCC: CRL-1718), DU4475 (ATCC: HTB-123), and NCIH522 (ATCC: CRL-5810) were cultured using standard techniques. Adherent cells were removed with PBS + 10 mM EDTA. 5 × 10⁷ cells were centrifuged at 800 g and pellets were resuspended in 10% neutral buffered formalin for 24 h at room temperature. Cells were subsequently centrifuged at 800 g, resuspended in 70% EtOH and stored at 4 °C until embedding into a paraffin block. PC3, A549, HeLa, and Pfeiffer cell pellets were embedded into a single block and designated as the control CPA. OAW28, CCFSTTG1, DU4475, and NCIH522 cell pellets were embedded into a single block and designated as the specificity CPA. The 60 cancer cell line FFPE CPAs were generated by Advanced Cell Diagnostics. Cell lines were fixed in 10% neutral buffered formalin for 24 h at 27 °C and then embedded into a paraffin block.

Caldwell C., Rottman J.B., Paces W., Bueche E., Reitsma S., Gibb J., Adisetiyo V., Haas M.S., Heath H., Newman W., Baum J., Gianani R, & Kagey M.H. (2021). Validation of a DKK1 RNAscope chromogenic in situ hybridization assay for gastric and gastroesophageal junction adenocarcinoma tumors. Scientific Reports, 11, 9920.

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Human Brain Astrocyte Acquisition and Characterization

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Human brain astrocytes (HBA) were purchased from Neuromics (Edina, MN), and iCell astrocytes from Cellular Dynamics International (Madison, WI), while the astrocytoma cell line CCF-STTG1 and HEK293 cells were both purchased from ATCC (Manassas, VA). A summary of cell lines included in the different experiments is specified in Table S7.

Lundin A., Delsing L., Clausen M., Ricchiuto P., Sanchez J., Sabirsh A., Ding M., Synnergren J., Zetterberg H., Brolén G., Hicks R., Herland A, & Falk A. (2018). Human iPS-Derived Astroglia from a Stable Neural Precursor State Show Improved Functionality Compared with Conventional Astrocytic Models. Stem Cell Reports, 10(3), 1030-1045.

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Glioma Cell Line Treatment with Inhibitors

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The NHA cell line was purchased from the Lonza group and cultured with Clonetics medium and reagents. Human glioma cell lines T98G, U87MG, A172, U251, and CCF-STTG1 were purchased from the ATCC and cultured according to the guidelines recommended by the ATCC. All cells were maintained at 37 °C with 5% CO₂. For drug treatment of cells, the mTOR inhibitor rapamycin (MedChemExpress, HY-10219) and PDK1 inhibitor OSU-03012 (Selleck, S1106) were used.

Li M., Cheng J., Ma Y., Guo H., Shu H., Huang H., Kuang Y, & Yang T. (2020). The histone demethylase JMJD2A promotes glioma cell growth via targeting Akt-mTOR signaling. Cancer Cell International, 20, 101.

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Cell culture and polyamine depletion

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CHO-K1, CCF-STTG1, HepG2, HEK293, Caco-2, MCF7 and PANC-1 were purchased from ATCC. ATDC5 were purchased from RIKEN Cell Bank, in Japan. U-2OS, HCT116, A549 and Neuro-2a were kindly supplied by Dr. Murai (The Jikei University School of Medicine, Japan), Dr. Fukumoto (Chiba University, Japan), Dr. Nakamura (Chiba University, Japan) and Dr. Kitagawa (Kobe Pharmaceutical University, Japan), respectively. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin G and 50 units/ml streptomycin in an atmosphere of 5% CO₂/95% air at 37 °C. A three-fold greater number of cells were cultured in DMEM medium in the presence of DFMO for 3 days to make polyamine-depleted cells. The concentration of DFMO used in this study was listed in Supplementary Table S8.

Imamura M., Higashi K., Yamaguchi K., Asakura K., Furihata T., Terui Y., Satake T., Maegawa J., Yasumura K., Ibuki A., Akase T., Nishimura K., Kashiwagi K., Linhardt R.J., Igarashi K, & Toida T. (2016). Polyamines release the let-7b-mediated suppression of initiation codon recognition during the protein synthesis of EXT2. Scientific Reports, 6, 33549.

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Diverse Astrocytoma Cell Lines for Research

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In the present study, four human astrocytoma cell lines of differing grades were used: CCF-STTG1 (grade IV, astrocytoma), SW1783 (grade III, astrocytoma), SW1088 (astrocytoma) and CHLA-03-AA (anaplastic astrocytoma). All cell lines were purchased from ATCC^®. SW1783 and SW1088 cells were grown as a monolayer in ATCC-formulated Leibovitz's L-15 medium (cat. no. 30-2008; ATCC) supplemented with 10% FBS and 50 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA). These two cell lines were grown at 37˚C in 100% air. For CCF-STTG1 and CHLA-03-AA cells, ATCC-formulated RPMI-1640 medium (catalog no. 30-2001; ATCC) supplemented with 10% FBS and 50 µg/ml streptomycin was used. These cells were grown at 37˚C with 5% CO₂. Prior to each experiment, the cells were detached using 0.25% trypsin with 0.02% EDTA (Sigma-Aldrich; Merck KGaA).

Choromanska A., Kulbacka J., Saczko J, & Surowiak P. (2020). Effect of diallyl disulfide and garlic oil on different human astrocytoma cell lines. Biomedical Reports, 13(4), 32.

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MDM2 Overexpression in Cancer Cells

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U2OS, SJSA-1, H1299 and CCF-STTG1 cells were purchased from ATCC. LS141 cells were a generous gift from Dr Jonathan Fletcher. Trp53^−/− and Trp53^−/−Mdm2^−/− MEFs were a generous gift from Carol A. Prives. Experiments in MEFs were carried out at matched cell passage numbers. The pCMV-Flag-Mdm2^WT expression plasmid expresses Flag-tagged, full-length human Mdm2 under the control of the CMV promoter. The pCMV-Flag-Mdm2^C464A expression plasmid was generated by site-directed mutagenesis using the pCMV-Flag-Mdm2^WT construct as a template. The recombinant, bicistronic Adeno-Mdm2-GFP adenovirus encodes human Mdm2 and green fluorescent protein, both of which are expressed by their respective CMV promoters. For establishment of stable Mdm2-overexpressing clones, U2OS cells were co-transfected with either empty pCDNA3, pCMV-Flag-Mdm2^WT, or pCMV-Flag-Mdm2^C464A and pBabe-Puro. Clones were selected in 1 µg/ml puromycin (Sigma, St. Louis, MO, USA).

Senturk J., Bohlman S, & Manfredi J. (2017). Mdm2 selectively suppresses DNA damage arising from inhibition of topoisomerase II independent of p53. Oncogene, 36(44), 6085-6096.

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Bat and Human Cell Culture Protocols

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Bat primary or immortalized cells (Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultia Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT) generated in our laboratory were all cultured in DMEM/F12 with 15% FBS [26 (link)]. Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS [36 (link)]. Human cells (Hep-2, A549, Calu-3, H292, Caco-2, Huh-7, Hela, HEK-293T, RD, CCF-STTG1, THP-1), bat lung epithelial cells (Tb1-Lu), and other mammalian cells (LLC-ML2, Vero, BHK21, MDCK, FK, SIEC, NIH-3T3, V79) originated from American Type Culture Collection (ATCC, www.atcc.org accessed on 18 September 2021) were maintained according to the recommendations.

Wang N., Luo C.M., Yang X.L., Liu H.Z., Zhang L.B., Zhang W., Li B., Zhu Y., Peng C., Shi Z.L, & Hu B. (2021). Genomic Characterization of Diverse Bat Coronavirus HKU10 in Hipposideros Bats. Viruses, 13(10), 1962.

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Glioma Cell Line Culture Protocol

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We used 8 glioma cell lines, namely U87MG, A172, LN405, SW1783, T98G, SW1088, CCF-STTG-1, and Gos-3. Cell lines U87MG, A172, and T98G were purchased from the European Collection of Cell Cultures (Salisbury, Wiltshire, UK). Cell lines SK-PN-DW, CCF-STTG1, SW1088, and SW1783 were purchased from the American Type Culture Collection (Manassas, VA, USA). Cell lines LN405 and Gos-3 were obtained from the Deutsche Sammlung Von Mikroorganismen und Zellkulturen (Braunschweig, Germany). Cell lines were cultured in RPMI L-Glutamax medium (GIBCO-BRL, Gaithersburg, MD, USA), supplemented with 10 % fetal bovine serum (FBS), 10 % non-essential amino acids (NEAs), 1 % penicillin, and 0.1 % amphotericin B. Cell lines were maintained in a 37 °C incubator with the supply of 5 % CO₂. Sub-culturing of cells was performed after 80 % confluence with the help of trypsin/EDTA 1X.

Shahi M.H., Zazpe I., Afzal M., Sinha S., Rebhun R.B., Meléndez B., Rey J.A, & Castresana J.S. (2014). Epigenetic regulation of human hedgehog interacting protein in glioma cell lines and primary tumor samples. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, 36(4), 2383-2391.

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