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【ES-2人卵巢透明细胞癌细胞 】下面是细胞株培养的具体无菌技术要点:
1. 实验开始前,无菌室和无菌操作台需要紫外光照射30到60分钟完全灭菌,用70%ethanol擦拭无菌操作台面,并且打开无菌操作台风扇运行10分钟之后,才可以进行实验操作。每次操作只能处理一株细胞株,即使培养基完全相同也不可共用培养基,以避免细胞间污染或失误混淆。实验完成后,请将实验物品拿出工作台,用70%ethanol再次擦拭无菌操作台面。操作间隔应该让无菌操作台运转10分钟到15分钟后,才能进行下一个细胞株的操作。
2. 无菌操作工作的区域应保持清洁和宽敞,必要物品(试管架、吸管、吸取器或吸管盒等)可以暂时放置,其它实验用品使用完毕后应移出,有利于空气流通。实验用品需70%ethanol擦拭后方可带入无菌操作台内。实验操作过程应在台面的中央无菌区域内完成,请勿在边缘非无菌区域进行操作。
3. 小心取用无菌实验物品,避免细菌污染。请勿触碰吸管尖头部和容器瓶口处,也不要在打开的容器正上方进行操作。容器打开后,请用手夹住瓶盖并轻握瓶身,倾斜大约45°角取用,尽量不要将瓶盖口朝上放置于桌面。
4. 工作人员应当首先注意自身安全,必须穿戴齐实验衣和实验手套后才能进行实验。对于病毒感染或是来自人类的细胞株应当特别小心操作,并选择适当等级的无菌操作台进行(至少是Class II)。操作过程中,应避免引起aerosol的产生,小心有毒性药品,例如DMSO和TPA等,并避免针头的伤害等等。
5. 定期检测下列项目:
5.1. CO2钢瓶的CO2压力
5.2. CO2培养箱的CO2浓度、温度、和水盘是否有污染(水盘的水需用无菌水,每周定时更换)。
5.3. 无菌操作台内的airflow压力,定期更换紫外线灯管和HEPA过滤膜,预滤网(300小时/预滤网,3000小时/HEPA)。
6. 水槽内可添加消毒剂(Zephrin 1:750),需定期更换水槽中的水。
【ES-2人卵巢透明细胞癌细胞 】齐一生物友情提醒,本产品仅供科研使用,不得用于临床及诊断使用!
一细胞说明,具体详细请见细胞附带说明书
二、客户自备试剂
1、PBS
2、 2、RPMI-1640 培养基(不含 Hepes)+ 10%胎牛血清+ 1%双抗
3、0.25% (W/V)胰蛋白酶(含 0.02% EDTA)
三、细胞背景
1、生长方式:贴壁
2、种属:Homo sapiens 人
四、培养条件
1、培养基:RPMI-1640 培养基(不含 Hepes)+ 10%胎牛血清+ 1%双抗
2、温度:37.0°C
3、气体:空气 95%,CO2 5%
【ES-2人卵巢透明细胞癌细胞 】五、培养方法
收到细胞后,在倒置显微镜下观察整个细胞生长情况:
如果细胞未长满,用 75%酒精喷洒整个瓶消毒后放到超菌台内,严格无菌操作,打开细胞培养瓶,留10ml 培养液继续培养。如果细胞已长满(达 80-90%)。即可进行传代,具六、体步骤如下:
1,弃去培养液,用PBS洗 1-2次。
2,向瓶内加入 1.0-2.0ml 胰蛋白酶液,在倒置显微镜下观察细胞消化情况,若细胞大部分变圆,迅速拿回操作台,吸取胰蛋白酶,加含有 6ml 含 10%血清的培养液,轻轻吹打细胞。
3,加入等量的的培养液,轻轻吹打混匀后吸出一半,分到新的培养
4,传代比例:1:2-1:3
七、常见问题及解决方案
1、培养瓶有破裂,培养液有漏液:细胞极大可能会污染,所以我们会及时安排帮老师解决。
2、细胞漂浮:培养瓶不开封,瓶口酒精擦拭后平躺放置在培养箱。次日观察,如细胞大部分又贴回瓶底,表明细胞活力正常,剩余漂浮的细胞可以去掉,留 10ml 培养液培养观察,细胞生长至汇合度 80%,进行消化传代;如细胞还是不贴壁,将细胞离心收集转到新培养瓶,原培养瓶加部分培养液继续培养,中间注意观察,我们的技术人员会一直跟踪指导,直到问题解决。
细胞培养代理品牌
| BD 产品涉及分子生物学、细胞生物学、免疫学和蛋白质组学等诸多领域,旗下拥有Clontech 、Discovery labware 、Pharmingen 、Falcon 等质品牌,值得称赞的是每一个品牌在其所在的领域都是其中的佼佼者。 |
细胞培养代理品牌
| GIBCO 作为细胞培养的金字招牌,是在此领域的领导者,现在是Invitrogen的一部分。Gibco可以提供最为广泛的品种以满足用户不同的需求,每一种产品都通过最为严格的品质管理。同时,Gibco也可按客户的要求对培养基的配方进行改动,或完全按客户的配方进行生产。 |
【ES-2人卵巢透明细胞癌细胞 】齐一生物科技(上海)有限公司是一家专业生产和销售各种生化试剂,细胞株、原代细胞、菌种、医药中间体,标准品和对照品的大型化学科技公司. 齐一生物销售:021-6034 8496;181214 53965;173021 04490;Web:www.qiyibio.com
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DML:
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