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AT1A/AT1B Double Knockout Immortalized Mouse Renal Proximal Tubule

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AT1A/AT1B Double Knockout Immortalized Mouse Renal Proximal Tubule

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AT1A/AT1B Double Knockout Immortalized Mouse Renal Proximal Tubule Cell Line

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	Print Version
Conditions of Sale	This Product is distributed under license from Case 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	C57B/6 mouse with AT_1A/1B^-/-genotype
SourceOrgan	Kidney
Growth Properties	Adherent
Morphology	Cobblestone
Description	The proximal tubule is part of the nephron duct system in the kidney, involved in regulating the renin-angiotension system. Cells of this region aid in regulating sodium and volume homeostasis as well as regulating the systemic blood pressure. The protein Angiotension II (Ang II) and its receptors AT₁ and AT₂ are some of the most studied proteins in this system. Even so, there is an incomplete understanding of the AT₁ receptors AT_1A and AT_1B. The AT1B Knockout Immortalized Mouse Renal Proximal Tubule Cell Line 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. When paired up with the wild type (Cat. No. T0624) and other angiotensin receptor (Ang II)-deficient cell lines AT_1A^-/- (Cat. No. T0626), AT_1B^-/- (Cat. No. T0627) and AT₂^-/- (Cat. No. T0625), these cell lines represent valuable tools to study fluid and electrolyte transportation in the proximal tubule region.
Procedure Overview
Recommended Seeding Density	Thaw entire contents into an appropriate T25 flask as specified in the Propagation instructions.
Applications	For Research Use Only
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. Grow cells on 5% gelatine and 10 mg/mL laminin (diluted 1:1) coated 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.
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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.
Disclaimer	1. The CoA for this product (provided upon request) verifies the cell-type specific gene expression via RT-PCR only. All test parameters provided in the CoA are conducted using abm's standardized culture system and procedures. The stated values may vary under the end-user's culture conditions. Please verify that the product is suitable for your studies by referencing published papers. All sales are final. 2. We strongly recommend live cell shipments for ease of cell transfer and this option can be requested at the time of ordering. Please note that the end-user will need to evaluate the feasibility of live cell shipment by taking into account the final destination's temperature variation and its geographical location. In addition, we thoroughly test our cell lines for freeze-thaw recovery. If frozen cells were received and not recovered in your lab under the exact, specified conditions (using recommended culture vessel, media, additional supplements, and atmospheric conditions), a live cell replacement is possible at a cost (plus shipping). 3. All of abm's cell biology products are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its cell biology product(s) in therapeutic/diagnostic application(s). Please contact a technical service representative for more information. 4. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature and provided for informational purposes only. abm does not warrant that such information has been shown to be accurate.