MCF-7细胞的培养传代.doc

1. MCF-7细胞的培养传代 细胞株购自武汉中国典藏物培养中心（美国ATCC株亚型），培养基是MEM， 10％的小牛血清（购自杭州四季青公司）。 传代要看细胞数的多少，一般来说10％的细胞，要5天左右，如果90％融合的细胞一传2，3天子代细胞即可传代。传代：偶用的是25ml培养瓶，吸出培养基，6mlpbs冲洗3次，加入0.25％的胰酶2－3ml，胰酶均匀盖满瓶壁，消化2－5min左右（可以同时振摇），细胞缩起变圆，浮起，加入培养基2－3ml，不用太精确，吹打成单细胞悬液分瓶即可。室温下消化即可，因为是成纤维细胞，较为耐受胰酶，消化时间稍长亦可，要注意mcf-7细胞是一种肿瘤细胞，生长规律性差，很容易不均匀，要注意吹打的充分性。ATCC Organ: mammary gland; breast Cellular products: insulin-like growth factor binding proteins (IGFBP) BP-2; BP-4; BP-5 Complete growth medium: The base medium for this cell line is ATCC-formulated Eagles Minimum Essential Medium, Catalog No. 30-2003. To make the complete growth medium, add the following components to the base medium: 0.01 mg/ml bovine insulin; fetal bovine serum to a final concentration of 10% .Atmosphere: air, 95%; carbon dioxide (CO2), 5%Temperature: 37.0°C Subculturing： 1. Remove culture medium to a centrifuge tube. 2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin - 0.53 mM EDTA (0.015%, w/v) 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 37C to facilitate dispersal. 4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting. 5. Transfer the cell suspension to the centrifuge tube with the medium and cells from step 1, and centrifuge at approximately 125 x g for 5 to 10 minutes. Discard the supernatant. 6. Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. 7. Incubate cultures at 37C. Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:6 is recommendedMedium Renewal: 2 to 3 times per week Freeze medium: Complete growth medium supplemented with 5% (v/v