209/MDCT cell line細胞株
209/MDCT (ATCC CRL-3250)Mus musculus, mouseDistal convoluted tubule cellsOrganism  Mus musculus, mouseTissue  kidneyCell Type  Distal convoluted tubule cellsProduct Format  frozenMorphology  epithelial-likeCulture Properties  adherentBiosafety Level  2  [Cells contain SV-40 viral DNA sequences]Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.Age  44-55 daysGender  maleApplications  Useful for studies of Na transport, implications of Gitelman’s syndrome, NaClcotransporter, ENaC, PTH signaling, calcium transport Receptor Expression  NaCl cotransporter (SLC12a3), expressed & PTH receptor, expressedComments  209/MDCT is a continuous line of distal convoluted tubule (DCT) cells expressing a phenotype including NaCl cotransporter (Slc12a3) and PTH receptors.  The DCT is a primary site for regulation of blood pressure by controlling NaCl absorption and for calcium homeostasis by parathyroid hormone.  This cell line is of considerable use for studies of hormonal and pharmacological regulation of fundamental ion transport processes and their underlying mechanisms. Complete Growth Medium  The base medium for this cell line is a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium with 1 g/L glucose and 1mM sodium pyruvate (Life Technologies, Catalog No. 11885) and Ham’s F-12 Nutrient Mix (Life Technologies, Catalog No. 11765). To make the complete growth medium, add the following component to the base medium:    fetal bovine serum to a final concentration of 5%Subculturing  Note: Subculture before cultures become 100% confluent. Cells can detach from flask when over-confluent.Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.     Remove and discard culture medium.    Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) or 0.05% (w/v) Trypsin - 0.02% EDTA solution to remove all traces of serum which contains trypsin inhibitor.    Add 1.0 to 2.0 mL of 0.05% Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).    Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37.0°C to facilitate dispersal.    Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.    Transfer cell suspension to a centrifuge tube and spin at approximately 125 xg for 5 to 10 minutes. Discard supernatant.    Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels.Subcultivation Ratio: A subcultivation ratio of 1:10 to 1:20 is recommendedMedium Renewal: 2 to 3 times per weekSeeding Density: 5.0 x 103 to 1.0 x 104 cells/cm2Cryopreservation  Freeze medium: complete growth medium, 70%;  FBS, 20%; DMSO, 10%Storage temperature: liquid nitrogen vapor phaseCulture Conditions  Temperature: 37°C;Atmosphere: air, 95%; carbon dioxide (CO2), 5% Year of Origin  1998References  Gesek FA, et al. Mechanism of calcium transport stimulated by chlorothiazide in mouse distal convoluted tubule cells. J. Clin. Invest. 90(2): 429-438, 1992. PubMed: 1322939Pizzonia JH, et al. Immunomagnetic separation, primary culture, and characterization of cortical thick ascending limb plus distal convoluted tubule cells from mouse kidney. In Vitro Cell Dev. Biol. 27A(5): 409-416, 1991. PubMed: 1649164
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