Immortalized Mouse Renal Proximal Tubule Cells

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Conditions of Sale	This Product is distributed under license from CAS:e Western Reserve University and may be used for internal research purposes only. The Licensed Products may not be used for clinical or diagnostic testing of persons and or animals.
BioSafety Level	II
Organism	Mus musculus
Source Organ	Kidney
Growth Properties	Adherent
Morphology	Cobblestone
Recommended Seeding Density	Thaw entire contents into an appropriate T25 flask as specified in the Propagation instructions.
Applications	For Research Use Only
Immortalization Method	Isolated from transgenic mouse carrying a temperature-sensitive simian virus 40 tumor antigen (tsA58)
Description	The renal proximal tubule is part of the nephron duct system in the kidney, playing important roles in functions like absorption and secretion of molecules in the renal system. The Immortalized Mouse Renal Proximal Tubule Cells is a conditionally immortalized mouse renal proximal tubule cells isolated from the mouse harboring thermolabile mutation (tsA58) of the simian virus 40 large T antigen. The functional expression of the SV40 large T antigen is induced by culturing the cells in vitro in medium containing IFN γ at a temperature permissive (33°C). At a non-permissive temperature (37°C-39°C), the cells cease to proliferate. These cells retain competent transepithelial electrolyte transport and short-circuit current activities. When paired up with angiotensin receptor (Ang II)-deficient cell lines AT1A-/-(Cat. No. T0626), AT1B-/-(Cat. No. T0627), AT1A-/-AT1B-/-(Cat. No. T0628) and AT2-/-(Cat. No. T0625), these cell lines represent valuable tools to study fluid and electrolyte transportation in the proximal tubule region.
Procedure Overview
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Propagation	Use of PriCoat? T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Grow cells in ECM-coated culture vessels with the following conditions. The base medium for this cell line is Prigrow IV medium available from ABM (TM004). To make the completed growth medium, add the following components to the base medium: 5% fetal bovine serum (TM999), 10nM aldosterone, 50μM L-ascorbic acid 2-phosphate, 4μg/mL dexamethasone, 10ng/mL epidermal growth factor, 5μg/mL insulin, 20nM sodium selenite (Na2SeO3), 5μg/mL transferrin, 1nM L-3,3’,5-triiodothyronine (T3), 10 U/mL recombinant mouse interferon gamma (IFN-γ) and 1% Penicillin/Streptomycin (G255). Atmosphere: air: 95%, CO?: 5%; Temperature: 33.0°C. Use of PriCoat? T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. To differentiate the cells, grow the cells at 37°C-39°C in the absence of IFN-γ.
Preservation	1. Freeze Medium: Complete growth medium with 20% FBS and 10% DMSO. 2. Storage Temperature: Liquid nitrogen vapour phase.
Quality Control	(1) RT-PCR was performed to confirm AT receptor genotype. (2) Electrical conductivity was measured to compare immortalized cells relative to non-immortalized counterparts. (3) Immunostaining was performed to determine localization of proteins associated with epithelial cells and assess morphology. (4) Changes in short circuit current were quantified to compare immortalized cell cotransporters to non-immortalized ones. (5) Western blotting was performed to detect the presence of AT receptor proteins. (6) Live cell microscopy of AngII-mediated β-arrestin 2 translocation was performed to measure receptor functionality.
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