经常有小伙伴们跟我说,在做细胞培养实验中,老是出现很多问题,有时候还忘记看说明书了,有时候不知道该怎么操作和注意什么问题,现在,我们一起来看看细胞培养中的实验tips。
关于血清
血清的热灭活在免疫分析时可以灭活补体系统。在免疫学研究,培养ES细胞,昆虫细胞和平滑肌细胞时,推荐使用热灭活血清。热灭活是以往公认的操作步骤,并没有确凿的证据说明这样做是有益的。相反,对大部分细胞而言,GIBCO和HyClone都不推荐热灭活血清。因为加热可能使血清中的沉淀增加,使您误以为污染。
如何避免血清中沉淀物的产生?
1. 解冻血清时,请按照所建议的逐步解冻法(-20℃至4℃至室温),若血清解冻时改变的温度太大(如-20℃至37℃),实验显示非常容易产生沉淀物。并随时将之摇晃均匀,使温度及成分均一,减少沉淀的发生。
2. 请勿将血清置于37℃太久。若在37℃放置太久,血清会变得混浊,同时血清中许多较不稳定的成分也会因此受到损害,而影响血清的质量。
3. 血清的热灭活非常容易造成沉淀物的增多,若非必要,可以无须做此步骤。
4. 若必须做血清的热灭活,请遵守56℃,30分钟的原则,并且随时摇晃均匀。温度过高,时间过久或摇晃不均匀,都会造成沉淀物的增多。
培养基的使用
细胞培养基的储存条件是2-8摄氏度。如果细胞培养基不小心被冻,您应该融化培养基并观察是否有沉淀产生。如果没有沉淀产生,培养基可以正常使用,如果出现沉淀,可能只能丢弃这些培养基。
★贮存在冰箱中的瓶口已开的培养基,可能在放置几天后颜色变紫。这主要是由于在暴露到周围的CO2水平时,碳酸氢纳导致了pH值的上升。您可以在使用前松开瓶口,在CO2培养箱孵育培养基10-15分钟,来校正溶液的pH值(确定松开瓶口以保证气体交换)。
★当在无血清培养基中添加抗生素时,降低至少在有血清培养基中所使用浓度的50%。血清蛋白会结合和灭活一些抗生素。在无血清培养条件下,抗生素不被灭活,可能对于细胞达到毒性水平。
★一旦您在新鲜培养基中添加了血清和抗生素时,您应该在两到三周内使用它。因为一些抗生素和血清中的基本成分在解冻后就开始降解。
细胞培养添加剂
大部分添加物和试剂最多可以冻融3次,如果次数更多都会在包含蛋白的溶液引起一定水平的降解和沉淀,将会影响它的性能。
★贮存在4℃冰箱中的液体胰蛋白酶溶液只能使用一周。胰蛋白酶在4℃就可能开始降解,如果在室温下放置超过30分钟,就会变得不稳定。
细胞复苏
在订购的细胞到达后,应立即复苏,如果培养基还没有准备好,可将其放入液氮中,尽快复苏。千万不能放置在-20或-80℃的冰箱内。
★细胞复苏往往是比较容易出问题的步骤。请在订购细胞时就向供应商索取详细的复苏步骤,并仔细阅读。有些供应商就不推荐在复苏时离心,对此类细节,应特别留意。
暂时就写这些啦,小伙伴还有别的关于细胞培养方面的知识需要了解的,可以在底部评论区留言哦!
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