Protocol: NOTE: the cells should not be allowed to become confluent, subculture at 80% of confluence. Remove medium, and rinse with 0.25% trypsin-0.53mM EDTA solution. Remove the solution and add an additional 1 to 2 ml of trypsin-EDTA solution. Allow the flask to sit at room temperature (or at 37.0°C) until the cells detach. Add fresh culture medium, aspirate and dispense into new culture flasks.
Subcultivation Ratio: A subcultivation ratio of 1:6 to 1:8 is recommended
Medium Renewal: Every 2 to 3 days
本段摘自ATCC的4T1细胞说明，一个是不能让它聚团，一个是要用胰酶洗一次以后再消化，有的细胞洗一次之后消化效果就会好很多。
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