欧美mv日韩mv国产-国产午夜一级片-一区二区在线 | 国-韩国无码一区二区三区免费视频-黄色a免费-欧美黄色大片免费观看-人人综合网-国产成人a在线观看视频免费-国内精品久久久久久久影视-国产老头和老太xxxx视频-高h放荡受浪受bl-国产又爽又黄又不遮挡视频-国产偷国产偷亚洲高清人乐享

1. Remove and discard culture medium.
2. Briefly rinse the cell layer, twice, with 5 mL Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) or 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. Add 5 mL of 0.25% 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°C to facilitate dispersal.

4. Add 5 mL of complete growth medium and aspirate cells by gently pipetting.
5. Transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes. Discard supernatant.
6. Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new vessels. An inoculum of 7.0 X 1⁴ to 1.0 X 10⁵ viable cells/cm² is recommended.
7. Incubate cultures at 37°C.

Muszynski M, et al. Glucocorticoids stimulate the production of preprocalcitonin-derived secretory peptides by a rat medullary thyroid carcinoma cell line. J. Biol. Chem. 258(19): 11678-83, 1983. PubMed: 6311819

Birnbaum RS, et al. Identification of procalcitonin in a rat medullary thyroid carcinoma cell line. J. Biol. Chem. 259(5): 2870-4, 1984. PubMed: 6199352

Russo AF, et al. Neuronal properties of a thyroid C-cell line: partial repression by dexamethasone and retinoic acid. Mol. Endocrinol. 6(2): 207-18, 1992. PubMed: 1569964

Birnbaum RS, et al. Biosynthesis of calcitonin by a rat medullary thyroid carcinoma cell line. J. Biol. Chem. 261(2): 699-703, 1986. PubMed: 3941098

Durham PL, et al. Repression of the calcitonin gene-related peptide promoter by 5-HT1 receptor activation. J. Neurosci. 17(24): 9545-53, 1997. PubMed: 9391009

Wood JL, et al. Autoregulation of cell-specific MAP kinase control of the tryptophan hydroxylase promoter. J. Biol. Chem. 276(24): 21262-71, 2001. PubMed: 11283010

Viney TJ, et al. Regulation of the cell-specific calcitonin/calcitonin gene-related peptide enhancer by USF and the Foxa2 forkhead protein. J. Biol. Chem. 279(48): 49948-55, 2004. PubMed: 15385550

Park KY, et al. Control of the calcitonin gene-related peptide enhancer by upstream stimulatory factor in trigeminal ganglion neurons. J. Biol. Chem. 283(9):5 441-51, 2008. PubMed: 18167349