细胞实验技术-课堂PPT-第四讲.ppt

1.Cultured Cells Freezing 2.Seeding cells for CCK-8 assay, Mitotic Index and Transfection Methods in Cell Biology Freezing Cultured Cells Cultured Cells Freezing Objective: Serves as a backup supply for emergencies. Provides a more homogeneous population by minimizing culture aging and evolution. Less work — saves time and money. Principle: When the cooling process is rapid, intracellular ice crystals form before complete cellular dehydration has occurred. These ice crystals disrupt cellular organelles and membranes and lead to cell death during the recovery (thawing) process. Principle: Principle: When the cooling process is slow, free intracellular water is osmotically pulled from the cells resulting in complete cellular dehydration and shrinkage. The physical stresses of cellular shrinking may cause some damage resulting in irreparable membrane loss and cytoskeletal and organelle disruption. Damage may also be caused by the high concentrations of solutes in the remaining unfrozen extracellular medium. These solutes attack cells both externally and internally, resulting in membrane damage, pH shifts and general protein denaturation. 在不加任何条件下直接冻存细胞时，细胞内和外环境中的水都会形成冰晶，能导致细胞内发生机械损伤、电解质升高、渗透压改变、脱水、PH改变、蛋白变性等，能引起细胞死亡。如向培养液加入保护剂，可使冰点降低。在缓慢的冻结条件下，能使细胞内水份在冻结前透出细胞。贮存在-130℃以下的低温中能减少冰晶的形成。 目前常用的保护剂为二甲亚砜（DMSO）和甘油，它们对细胞无毒性，分子量小，溶解度大，易穿透细胞。 Reagents required for Cells Freezing : ? Medium; ? Pipetting devices; ? Sterile polypropylene conical tube, 50ml and 15ml; ? 2 ml cryovial; ? DMSO; ? Hemacytometer; ? Tabletopcentrifuge; ? -80°C freezer ? Liquid nitrogen container Protocol： (1) Trypsinize cells and harvest in the normal way and resuspend them in one half the volume that you want to end up with. In other words, use 2 ml per 100 mm dish you are using. The cells should be resuspended in DMEM containing 10% fetal calf serum. Protocol： (2) Count a 200 ul aliquot and determine the total cell number. From this, calculate the