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GeneChip analyses of cell lines harvested at different passages, and as xenografted tumors, indicated that PNECs express consistent features ex vivo and in vivo and share a remarkable degree of similarity with primary CR2-TAg prostate neuroendocrine (NE) tumors. 

PNECs express mAsh1 (mouse homolog proneural gene complex achaete-scute), a basic helix-loop-helix (bHLH) transcription factor essential for NE cell differentiation in other tissues. PNEC cell lines should be useful for genetic and/or pharmacologic studies of the regulation of NE cell proliferation, differentiation, and tumorigenesis.

• additives that are supplied with the kit (ATCC does not use gentamycin-amphotericin B)
• heat-inactivated fetal bovine serum (FBS) to a final concentration of 10%
• bovine pituitary extract (BPE) (Lonza/Clonetics, Inc., Catalog No. CC-4009) to a final concentration of 0.3%

Volumes used in this protocol are for 75 cm². flasks; proportionally reduce or increase amount of solutions for culture vessels of other sizes.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 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 2.0 to 3.0 mL of 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 6.0 to 8.0 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 poly-D-lysine and laminin coated culture vessels. An inoculum of 2 X 10⁴ to 5 X 10⁴ viable cells/cm² is recommended.
7. Incubate cultures at 37°C. subculture when cultures reach a cell concentration between 1 X 10⁵ and 2 X 10⁵ cells/cm²

Ippolito JE, et al. An integrated functional genomics and metabolomics approach for defining poor prognosis in human neuroendocrine cancers. Proc. Natl. Acad. Sci. 102(28):9901-9906, 2005. PubMed: 15998737

Hu Y, et al. RNA interference of achaete-scute homolog 1 in mouse prostate neuroendocrine cells reveals its gene targets and DNA binding sites. Proc. Natl. Acad. Sci. 101(15):5559-5564, 2004. PubMed: 15060276

Hu Y, et al. RNA interference of achaete-scute homolog 1 in mouse prostate neuroendocrine cells reveals its gene targets and DNA binding sites. Proc. Natl. Acad. Sci. 101(15):5559-5564, 2004. PubMed: 15060276