亚洲国产成人精品久久久国产成人一区二区三区综合区精品久久久中文字幕一区,国产a一级无码毛片一区二区三区,久久久无码,国产成人无码精品久免费,精品欧美国产一区二区三区不卡,国产成人一区二区三区影院,国产精品久久久久久,欧美日韩精品一区二区三区,欧美日韩在线精品一区二区三区激情福利综合,在线观看亚洲精品福利片,亚洲欧美日韩久久精品,亚洲欧美日韩国产成人精品,亚洲国产欧美日韩精品一区二区三区,欧美日韩国产成人高清视频

1. Remove and discard culture medium. Briefly rinse the cell layer with Ca⁺⁺/Mg⁺⁺ free Dulbecco's phosphate-buffered saline (D-PBS) (ATCC^® 30-2200) or 0.05% Trypsin – 0.02% EDTA for Primary Cells (ATCC^® PCS-999-003) solution to remove all traces of serum which contains trypsin inhibitor.
2. 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.
3. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
4. Transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
5. Discard supernatant. Resuspend the cell pellet in fresh growth medium.
6. Add appropriate aliquots of the cell suspension to new culture vessels. Incubate cultures at 37°C.

Subcultivation ratio: 1:5 to 1:20 is recommended.

Medium renewal: 2 to 3 times a week

Watson PA, et al. Context-dependent hormone-refractory progression revealed through characterization of a novel murine prostate cancer cell line. Cancer Res. 65(24): 11565-11571, 2005. PubMed: 16357166