Rle 6tn

Manufactured by American Type Culture Collection

Sourced in United States

The RLE-6TN is a laboratory equipment product designed for cell culture applications. It functions as a roller bottle incubator, providing a controlled environment for the cultivation and maintenance of cells in roller bottles. The product specifications and core functionality are provided without interpretation or extrapolation on intended use.

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27 protocols using rle 6tn

Evaluating EVs on Fungal Viability

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To test the effect of EVs on fungal viability, P. carinii (5 × 10⁷ nuclei) were inoculated into 48-well plates (Costar 3548, Corning, New York). Vehicle control or EV samples (2-µg protein equivalent) were added to the wells. Plates were incubated at 5% CO₂, 37°C. At 1, 3, 5, and 7 days, 100-µL samples were transferred to opaque white plates (USA Scientific, Ocala, FL) and assessed for ATP content using ATPlite (Perkin-Elmer, Waltham, MA).
Rat alveolar cell line, RLE-6TN (ATCC# CRL-2300; 4 × 10⁴ cells/well), was inoculated into opaque white plates (USA Scientific, Ocala, FL) and treated with 2-µg EVs. At 1, 3, and 5 days, samples were assessed for ATP content using the methods described above.

Sayson S.G., Ashbaugh A, & Cushion M.T. (2024). Extracellular vesicles from Pneumocystis carinii-infected rats impair fungal viability but are dispensable for macrophage functions. Microbiology Spectrum, 12(2), e03653-23.

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Modulation of Alveolar Epithelial Cells

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In vitro, rat type II alveolar epithelial cells line (RLE-6TN) was purchased from ATCC (Manassas, USA), and human lung cancer cell (A549) donated by teacher Shao, who works in the Second Affiliated Hospital of Dalian Medical University, all cells grew in DMEM medium (Hyclone, USA) supplemented with 9% fetal bovine serum (FBS; Gibco, USA), the cells were incubated at 37 °C with 5% CO₂. Cells were incubated with recombinant human TGF-β1 (100–21 C, PeproTech) or SFN (1 μmol/L, Sigma S6317)^{14 (link)} in some experiments.

Zhang Z., Qu J., Zheng C., Zhang P., Zhou W., Cui W., Mo X., Li L., Xu L, & Gao J. (2018). Nrf2 antioxidant pathway suppresses Numb-mediated epithelial–mesenchymal transition during pulmonary fibrosis. Cell Death & Disease, 9(2), 83.

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Rat AECs Treated with TGF-β1 and SFN

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Rat type II AECs (RLE-6TN) were purchased from ATCC (Manassas, USA), and grown in 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), and the cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂. In some experiments, the cells were incubated with recombinant human TGF-β1 (100–21 C, PeproTech) or SFN (1 μmol/L, Sigma S6317)20 (link)28 (link). Then, cell lysates were harvested for Western blot analysis.

Zhou W., Mo X., Cui W., Zhang Z., Li D., Li L., Xu L., Yao H, & Gao J. (2016). Nrf2 inhibits epithelial-mesenchymal transition by suppressing snail expression during pulmonary fibrosis. Scientific Reports, 6, 38646.

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Characterization of Rat Alveolar Epithelial Cells

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Rat type II alveolar epithelial cell line RLE-6TN was obtained from ATCC and maintained at 37 °C 5% CO₂ in DMEM (Invitrogen) with 4.5 g/l glucose and 2 mM L-glutamine, supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were tested periodically for mycoplasma contamination using the Universal Mycoplasma Detection Kit (ATCC). Generation and validation of stable p52 over-expressing (RLE-p52) and control empty vector (RLE-EV) cells are described elsewhere^{19 (link)}. RLE-EV and RLE-p52 cells were plated in 96 well plates for viability and BrdU incorporation measurements. 48 hours after plating, cells were incubated with BrdU for 4 hours, and BrdU incorporation was measured using the chemiluminescent BrdU Cell Proliferation ELISA (Roche). To ensure plating of equal numbers of cells, viability of RLE-EV and RLE-p52 cells was measured on the same day as a surrogate for cell number using the CellTiter-Glo Luminescent Cell Viability assay (Promega) according to the manufacturer’s protocol.

Saxon J.A., Yu H., Polosukhin V.V., Stathopoulos G.T., Gleaves L.A., McLoed A.G., Massion P.P., Yull F.E., Zhao Z, & Blackwell T.S. (2018). p52 expression enhances lung cancer progression. Scientific Reports, 8, 6078.

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LPS-Induced Alveolar Type II Cell Activation

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Alveolar type II cells (RLE-6TN; ATCC) were maintained in Dulbecco’s modified eagle’s medium (Hyclone, USA) containing 10% fetal bovine serum (Hyclone) and induced with 0, 1, 5, and 10 μg/mL LPS (Sigma-Aldrich) for 48 h to mimic septic ALI.

Ding F., Zhu J, & Hu Y. (2023). Circular RNA protein tyrosine kinase 2 aggravates pyroptosis and inflammation in septic lung tissue by promoting microRNA-766/eukaryotic initiation factor 5A axis-mediated ATP efflux. Acta Cirúrgica Brasileira, 38, e380323.

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Hypoxia and TGF-β Induced Cell Migration

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H358 cells (with epithelial characteristics) and immortalized rat alveolar type II cell lines RLE‐6TN were purchased from ATCC. CC2512 cells (human adult normal lung fibroblasts) were from Lonza (Basel, Switzerland). The Dox‐dependent gene expression system was applied to H358 cells carrying pTet‐On Advanced (H358ON).10, 11 Cells were treated with TGF‐β (2 ng/mL) for the indicated period. FG4592 (FG) at 30 μg/mL, CoCl₂ at 150 nmol/L, OA at 20 nmol/L, and/or Dox 1 μg/mL were also added to the cells for the indicated period. The cells were cultured under hypoxia (1% O₂) for the indicated periods using a hypoxic chamber (Wakenyaku Co. Ltd, Kyoto, Japan).19, 20 The in vitro scratch assay was also carried out.21

Ando A., Hashimoto N., Sakamoto K., Omote N., Miyazaki S., Nakahara Y., Imaizumi K., Kawabe T, & Hasegawa Y. (2019). Repressive role of stabilized hypoxia inducible factor 1α expression on transforming growth factor β‐induced extracellular matrix production in lung cancer cells. Cancer Science, 110(6), 1959-1973.

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Rat Alveolar Epithelial Cell Culture

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The rat AEC-II cell line RLE-6TN (ATCC, Manassas, VA, USA) was purchased and cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, US) and 1% penicillin/streptomycin (HyClone, South Logan, UT, USA), and maintained at 37 °C, 5% CO2 humidified incubator. For the model group, the cell was incubated in a high-oxygen environment (85% O₂, 5% CO₂) for 48 h.

Wu D., Bai D., Yang M., Wu B, & Xu W. (2024). Role of Sox9 in BPD and its effects on the Wnt/β-catenin pathway and AEC-II differentiation. Cell Death Discovery, 10, 20.

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Bleomycin-Induced Lung Epithelial Cell Injury

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The rat type II lung epithelial cell line RLE-6TN (ATCC Cell Bank); bleomycin (Zhejiang Hisun Pharmaceutical Co., Ltd., certificate number: National Medicine H20055883); Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α (Abcam, ab46070), KL-6 (Abcam, ab100772), and E-cadherin (Yaki Bio, China, IH-1519R); a reverse transcription kit (Takara, China); a qPCR kit (Takara, China); antibodies targeting KL-6, (ab84597, Abcam, 1:1,000), STAT3 (ab68153, Abcam, 1:1,000), phosphorylated signal transducer and activator of transcription 3 (p-STAT3, sc-293059, Santa Cruz, 1:1,000), α-smooth muscle actin (α-SMA, ab232784, Abcam, 1:1,000), and vimentin (ab92547, Abcam, 1:1,000); goat anti-rabbit IgG H&L (HRP) (ab97051, Abcam, 1:5,000), and an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit (Biyuntian Bio, C0009S) were used in this study.

Zuo Y., Liu J., Xu H., Li Y., Tao R, & Zhang Z. (2022). Pirfenidone inhibits cell fibrosis in connective tissue disease-associated interstitial lung disease by targeting the TNF-α/STAT3/KL6 pathway. Journal of Thoracic Disease, 14(6), 2089-2102.

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RLE-6TN Cell Line Culture

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RLE-6TN (ATCC CRL-2300) cell line was purchased from ATCC (Manassas VA, USA). Cells were cultured in HAM´s F12 (Gibco, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and supplemented with fetal bovine serum (FBS) (Gibco), penicillin (100 U/mL)/streptomycin (100 μg/mL) solution (Gibco), 2 mM L-glutamine (Gibco), 0.01 mg/mL bovine pituitary extract (Gibco), 0.005 mg/ mL insulin (Gibco), 2.5 ng/mL insulin-like growth factor (Gibco), 0.00125 mg/mL transferrin (Gibco), and 2.5 ng/ mL EGF (Gibco) and maintained at 37°C in a humidified 5% CO2 atmosphere.

Pintado-Berninches L., Montes-Worboys A., Manguan-García C., Arias-Salgado E.G., Serrano A., Fernandez-Varas B., Guerrero-López R., Iarriccio L., Planas L., Guenechea G., Egusquiaguirre S.P., Hernandez R.M., Igartua M., Luis Pedraz J., Cortijo J., Sastre L., Molina-Molina M, & Perona R. (2021). GSE4-loaded nanoparticles a potential therapy for lung fibrosis that enhances pneumocyte growth, reduces apoptosis and DNA damage. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 35(3).

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EMT Induction in Alveolar Type II Cells

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Alveolar type Ⅱ epithelial (RLE‐6TN) was obtained from ATCC (Rockville, Maryland, USA). The RLE‐6TN cells were cultured in 1640 medium (Hyclone) with 10% foetal bovine serum (FBS; Thermo Fisher Scientific), 1% penicillin/streptomycin (Beyotime) at 37°C with 5% CO₂ in a humidified atmosphere. For inducing EMT, the complete medium was replaced with serum‐free medium (containing 0.1% FBS), in which the cells were incubated with recombinant TGF‐β1 (10 ng/mL, PeproTech).15

Qian W., Cai X., Qian Q., Zhang W, & Tian L. (2020). Metastasis‐associated protein 1 promotes epithelial‐mesenchymal transition in idiopathic pulmonary fibrosis by up‐regulating Snail expression. Journal of Cellular and Molecular Medicine, 24(11), 5998-6007.

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