293T说明与培养方法.doc

293T (ATCCCRL-3216)DerivationThe 293T cell line, originally referred as 293tsA1609neo, is a highly transfectable derivative of human embryonic kidney 293 cells, and contains the SV40 T-antigen.CommentsThis cell line is competent to replicate vectors carrying the SV40 region of replication. It gives high titers when used to produce retroviruses. It has been widely used for retroviral production, gene expression and protein production.Culture MethodComplete Growth MediumThe base medium for this cell line is Dulbeccos Modified Eagles Medium (DMEM) (ATCC 30-2002). To make the complete growth medium, add the following components to the base medium: 10% Fetal Bovine Serum (heat inactivated) (ATCC 30-2020), 2mM L-glutamine (ATCC 30-2214), 1% Penicillin/Streptomycin.SubculturingVolumes used in this protocol are for 75 cm2flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.1. Remove and discard culture medium.2. Briefly rinse the cell layer with Ca+/Mg+ free Dulbeccos phosphate-buffered saline (D-PBS) or 0.05% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.3. Add 1.0 to 2.0 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. Add appropriate aliquots of the cell suspension to new culture vessels. Incubate cultures at 37C.Subcultivation ratio:A subcultivation ratio of 1:3 to 1:8 is recommended.Medium renewal:Every 2 to 3 daysCryopreservationFreeze medium:complete growth medium supplemented with 5% (v/v) DMSOStorage temperature:liquid nitrogen vapor phaseTemperature:37CAtmosphere:air, 95%; carbon dioxide (CO2)293T (ATCCCRL-3216)来源293T细胞系，最初引于293tsA1609neo，是一种可高度转染的衍生于人胚肾293细胞的细胞系，包含SV40 T-抗原。备注该细胞系可以复制包含SV40复制区域的质粒。用于产生逆病毒时可获得高滴度。该细胞系广泛用于逆病毒生产，基因表达和蛋白质生产。培养方法完全培养基此细胞系的基础培养基为ATCC配方的DMEM(货号30-2002)。配制完全生长培养基，在基础培养基中加入以下成分：10%的胎牛血清（热灭活）（ATCC 30-2020），2mM L-谷氨酰胺（ATCC 30-2214），1%的青霉素/链霉素。传代体积设定为75cm2烧瓶。如果是其他型号，请按体积增加或减少培养液。1. 除去并丢弃培养液。2. 用Ca2+/Mg2+free的（D-PBS）或者0.05%（w/v）胰蛋白酶-0.53mM EDTA溶液来去除所有包含胰蛋白酶抑制剂的血清残留。3. 加1.0到2.0 mL的胰蛋白酶-EDTA溶液到培养瓶，在倒置显微镜下观察细胞直到细胞层脱离（通常在515分钟）。 注: 为了避免聚集，在等待细胞脱落时不要通过拍打或者摇动培养瓶来晃动细胞。如果细胞难以脱离，可以放在37C加速脱离。