Htert rpe1

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HTERT-RPE1 is a cell line derived from human retinal pigment epithelial cells. It is immortalized through the introduction of the human telomerase reverse transcriptase (hTERT) gene. This cell line maintains the phenotypic characteristics of primary retinal pigment epithelial cells and can be used for various research applications.

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HTERT-RPE1 cells (40 citations) HTERT-RPE1 cells (CRL-4000; (2 citations) RPE1-hTERT human retinal pigment epithelial cells (2 citations)

75 protocols using htert rpe1

Cell Culture and Transfection Protocols

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hTERT-RPE-1, RCTE and HEK293T cells were purchased from ATCC (Manassas, VA, USA) hTERT-RPE-1, RCTE and GANAB^−/− cells were cultured in DMEM/F12 containing 10% fetal bovine serum, supplemented with penicillin and streptomycin. HEK293T cells were cultured in DMEM containing 10% fetal bovine serum, supplemented with penicillin and streptomycin.
For plasmid transfection, X-tremeGENE 9 or FuGENE 6 (Roche) was used, according to the manufacturer’s instructions. For siRNAs transfection, Lipofectamine RNAiMAX (Invitrogene) was used, according to the manufacturer’s instructions.

He K., Ma X., Xu T., Li Y., Hodge A., Zhang Q., Torline J., Huang Y., Zhao J., Ling K, & Hu J. (2018). Axoneme polyglutamylation regulated by Joubert syndrome protein ARL13B controls ciliary targeting of signaling molecules. Nature Communications, 9, 3310.

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Cultivation and Transfection of RPE1, HEK293T, and U2OS Cells

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Human telomerase immortalized retinal pigment epithelium cells (hTERT-RPE1, ATCC) were cultured with Dulbecco’s modified Eagle’s Medium DMEM/F12 50/50 medium (Pan Biotech) supplemented with 10% fetal bovine serum (FBS, Life Technologies) and1% penicillin-streptomycin (Gibco). Human embryonic kidney (HEK293T, ATCC) and osteosarcoma epithelial (U2OS, ATCC) cells were cultured with DMEM medium (Pan Biotech) supplemented with10% FBS and 1% penicillin-streptomycin. All cell lines were authenticated by Multiplex Cell Line Authentication (MCA) and were tested for mycoplasma by MycoAlert Mycoplasma Detection Kit (Lonza). U2OS cells were transfected with the plasmids using Lipofectamine 2000 and according to the manufacturer’s instructions (Thermo Scientific). HEK293T cells were transfected with the plasmids using 1 mg/ml polyethylenimine, MW 25 kDa (PEI). For microtubule depolymerization experiments, cells were treated with 5 μg/ml nocodazole (Sigma-Aldrich) or vehicle (dimethyl sulfoxide) for 1 h at 37°C. For cell synchronization experiments, 5 μm (+)-S-trityl-L-cysteine (STLC) (Alfa-Aesar) was used for 16 h at 37°C.

Batman U., Deretic J, & Firat-Karalar E.N. (2022). The ciliopathy protein CCDC66 controls mitotic progression and cytokinesis by promoting microtubule nucleation and organization. PLoS Biology, 20(7), e3001708.

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Cell Transfection Protocols Across Lines

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h-TERT-immortalized HA^{51 (link)}, SF126 cells^{52 (link)}, U87 (ATCC HTB-14), h-TERT-RPE-1 (ATCC CRL-4000), HEK293T (ATCC CRL-11268), U251 (Sigma # 09063001). Cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Sigma). Cells were transfected using Lipofectamine 2000 (Invitrogen) or the calcium phosphate method.

Frattini V., Pagnotta S.M., T.a.l.a., Fan J.J., Russo M.V., Lee S.B., Garofano L., Zhang J., Shi P., Lewis G., Sanson H., Frederick V., Castano A.M., Cerulo L., Rolland D.C., Mall R., Mokhtari K., Elenitoba-Johnson K.S., Sanson M., Huang X., Ceccarelli M., Lasorella A, & Iavarone A. (2018). A metabolic function of FGFR3-TACC3 gene fusions in cancer. Nature, 553(7687), 222-227.

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Engineered Retinal Pigment Epithelial Cells

Cited 3 times

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The hTERT-immortalized human retinal pigment epithelial cells (hTERT-RPE-1) were obtained from ATCC (Manassas, VA) and grown in DMEM/F12, 1:1 (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) medium supplemented with 10% foetal bovine serum (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA), penicillin/streptomycin (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA), and 0.01 mg/ml hygromycin-B (Millipore Sigma, Darmstadt, Germany). The cells were tagged with mEmerald-vimentin using the TALEN genome editing approach, which has been previously described^{29 (link)}. For imaging, the cells were placed into collagen as previously described^{21 (link)}. MV3 cells were obtained from Peter Friedl (MD Anderson Cancer Center, Houston TX). The MV3 cells were cultured in DMEM (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% foetal bovine serum (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37 °C and 5% CO₂. The cells were infected to express GEMs using a lentiviral construct from Addgene (Plasmid #116934^{22 (link)}). The cells were sorted by fluorescence activated cell sorting to purify the population of cells expressing GEMs.

Chakraborty T., Chen B., Daetwyler S., Chang B.J., Vanderpoorten O., Sapoznik E., Kaminski C.F., Knowles T.P., Dean K.M, & Fiolka R. (2020). Converting lateral scanning into axial focusing to speed up three-dimensional microscopy. Light, Science & Applications, 9, 165.

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Cell Culture Protocols for Cancer Research

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The A673 cell line was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and cultured in Dulbecco’s Modified Eagle Medium (DMEM, Cat. No. 044–29765, Fujifilm-Wako chemical) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1x Penicillin-Streptomycin Solution (Cat. No. 168–23191, Fujifilm-Wako chemical). The Seki cell line was established by Nojima et al. [20 (link)], purchased from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Cat. No. TKG 0725, Miyagi, Japan), and cultured in RPMI-1640 (Cat. No. 189–02025, Fujifilm-Wako chemical) with 10% FBS and 1x Penicillin-Streptomycin Solution. The NCR-EW2 cell line and SAOS-2 cell line (ATCC HTB-85) were cultured in RPMI-1640 with 10% FBS and 1x Penicillin-Streptomycin Solution. Human Embryonic Kidney cells 293 (HEK293) cells and hTERT RPE-1 (ATCC CRL-400), Lenti-X^™ 293T cell line (Cat. No. 632180, Takara-bio), and U-2 OS cell line were cultured in DMEM with 10% FBS and 1x Penicillin-Streptomycin Solution. Seki, NCR-EW2, and Lenti-X293T cells were spread onto a 0.1% gelatin-coated dish.

Kitagawa T., Kobayashi D., Baron B., Okita H., Miyamoto T., Takai R., Paudel D., Ohta T., Asaoka Y., Tokunaga M., Nakagawa K., Furutani-Seiki M., Araki N., Kuramitsu Y, & Kobayashi M. (2022). AT-hook DNA-binding motif-containing protein one knockdown downregulates EWS-FLI1 transcriptional activity in Ewing’s sarcoma cells. PLoS ONE, 17(10), e0269077.

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Cell Culture and Transfection Protocols

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Human cervical carcinomas HeLa cells, human breast adenocarcinoma MCF7 cells and RPE1 (hTERT-RPE1) cells were purchased from ATCC. The validation of the identity of HeLa, MCF7 and RPE1 cells was determined by Tianjin Weikai Bioeng LTD (China, Tianjin). HeLa and MCF7 cells were cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum (FBS) (HyClone). RPE1 cells were cultured in DMEM: F-12 (1:1) (HyClone) containing 10% FBS. All cells were cultured at 37°C in 5% CO₂. Plasmid/siRNA transfection was conducted with Lipofectamine 3000 and/or RNAiMAX Reagent (Life Technologies Inc) according to manufacturer's protocol. In brief, HeLa, MCF7 and RPE1 cells were plated in 96-well plates (3000 cells/well), 24-well plates (2×10⁴ cells/well) or 6-well plates (1×10⁵ cells/well) for 16 h at 37°C before transfection. Then, cells were incubated with indicated plasmids/siRNAs plus transfection reagent mixture in medium for 24 h and then changed into fresh medium. Cycloheximide (CHX) (Aladdin) and Actinomycin D (ActD) (Byotime) were used for cell treatment at final concentration of 10 μM CHX and 5 nM ActD at indicated times as previously described [52 (link)].

Dong Z., Zhu C., Zhan Q, & Jiang W. (2017). The roles of RRP15 in nucleolar formation, ribosome biogenesis and checkpoint control in human cells. Oncotarget, 8(8), 13240-13252.

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Cell Line Authentication and Mycoplasma Testing

Cited 2 times

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The HCT116 cell line and its mutant derivatives (31 (link),37 (link)) were generously provided by Drs. Eric Hendrickson and Alex Sobeck at the University of Minnesota. These lines have been authenticated by these researchers in house. The H1299, U2OS, HeLa, hTERT-RPE1, BJ-5ta, HDFn and IMR90 cell lines were purchased from ATCC between 2017–2018 and were cultured less than 8 weeks from frozen stocks of early passages (p2–4) for this study. All cell culture media were purchased from Sigma-Aldrich, supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals), and Penicillin-streptomycin (Thermo Fisher). Mycoplasma testing has not been performed on these cell lines. However, HCT116 and U2OS cells were cultured in McCoy’s 5A (M8403) additionally supplemented with Plasmocin™ (mycoplasma elimination reagent, Invivogen) and GlutaMax™ (Thermo Fisher). H1299 cells were cultured in RPMI-1640 (R8758). RPE1 cells were cultured in DMEM/F12 (D6421) additionally supplemented with GlutaMax™ and 0.01% hygromycin B (Sigma). BJ cells were cultured in a 4:1 media mixture of DMEM (D6429) and Medium 199 (M4530) additionally supplemented with 0.01% hygromycin B. HDFn and Hela cells were cultured in DMEM (D6429). IMR90 cells were cultured in MEM (51416C) additionally supplemented with GlutaMax™.

Graber-Feesl C.L., Pederson K.D., Aney K.J, & Shima N. (2019). Mitotic DNA synthesis is differentially regulated between cancer and non-cancerous cells. Molecular cancer research : MCR.

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Rapid Degradation of COG4 in RPE1 and HEK293T Cells

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hTERT RPE1 (retinal pigment epithelial) and HEK293T cells were purchased from ATCC. hTERT RPE1 COG4 KO cells were described previously.^{29 (link)} Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing Nutrient mixture F-12 (DMEM/F12, Corning 10–092-CV) supplemented with 10% fetal bovine serum (Atlas Biologicals, CF-0500-A). Cells were incubated in a 37°C incubator with 5% CO₂ and 90% humidity.
For rapid COG4 degradation, a stock solution of 0.5 M Indole-3-acetic acid sodium salt (auxin, IAA, Sigma # I5148) was prepared in water and stored in a frozen aliquot. Time course treatment of cells was performed with 500 μM IAA for 0.5, 1, 2, 24, and 48 h at 37°C. The cells without auxin treatment were considered as untreated control.

Sumya F.T., Pokrovskaya I.D., D'Souza Z, & Lupashin V.V. (2022). Acute COG complex inactivation unveiled its immediate impact on Golgi and illuminated the nature of intra‐Golgi recycling vesicles. Traffic (Copenhagen, Denmark), 24(2), 52-75.

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Cell Culture Conditions Across Cell Lines

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The human cell lines: DU145 (DSMZ ACC 261), MDAMB231 (DSMZ ACC 732), Panc-1 (DSMZ ACC 783) and hTERT RPE1 (ATCC CRL-4000) were grown in Dulbecco’s MEM medium supplemented with 10% fetal bovine serum. SHYS5Y (DSMZ ACC 209), HeLa (DSMZ ACC 57) and MCF-7 (DSMZ ACC 115) were grown in RPMI medium supplemented with 10% fetal bovine serum, MDAMB435S (ATCC HTB-129) were grown in RPMI medium supplemented with 15% fetal bovine serum and MiaPaca-2 (DSMZ ACC 733) cells were grown in DMEM-F12 medium supplemented with 10% fetal bovine serum. All media were purchased from Thermo Fisher Scientific (Schwerte, Germany), and bovine serum from Biochrom (Berlin, Germany) or PAA (GE Healthcare, Freiburg, Germany). All cell lines were incubated under a humidified atmosphere of 95% air/5% CO₂ at 37 °C.

Hernández-Reséndiz I., Pacheu-Grau D., Sánchez A, & Pardo L.A. (2020). Inhibition of Kv10.1 Channels Sensitizes Mitochondria of Cancer Cells to Antimetabolic Agents. Cancers, 12(4), 920.

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Rapid COG4 Degradation in RPE1 Cells

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hTERT RPE1 (retinal pigment epithelial) and HEK293T cells were purchased from ATCC. hTERT RPE1 COG4 KO cells were described previously.²⁹ Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing Nutrient mixture F‐12 (DMEM/F12, Corning 10–092‐CV) supplemented with 10% fetal bovine serum (Atlas Biologicals, CF‐0500‐A). Cells were incubated in a 37°C incubator with 5% CO₂ and 90% humidity.
For rapid COG4 degradation, a stock solution of 0.5 M Indole‐3‐acetic acid sodium salt (auxin, IAA, Sigma # I5148) was prepared in water and stored in a frozen aliquot. Time course treatment of cells was performed with 500 μM IAA for 0.5, 1, 2, 24, and 48 h at 37°C. The cells without auxin treatment were considered as untreated control.

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