Hello, colleagues.
This is my first time I am trying to grow a cell culture from ATCC. So it is Tera-1 (HTB-105) cells and what they recommend on their site is that the medium has to be the McCoy 5A (the one that the y recommend is http://www.atcc.org/products/all/30-2007.aspx but what I have bought for that is http://www.lifetechnologies.com/order/catalog/product/16600082) and it has just to be supplemented with 15% FBS - that's all they say...
In fact, after browsing some papers concerning that subject I have discovered that almost everyone does that his own way.
So now the question is basically, if I need to add penstrep to the above mentioned media, for example? Do I need HEPES in it? It looks like L-Gln is already there so I don't need it.
Or I don't need all these and just add FBS to a final concentration of 15%?
Would you be so kind to clarify a little this question for me?
Many thanks to you in advance! Just don't want to spoil that vial that arrived this morning :)
It is ok to use McCoy's 5A from Invitrogen for your cell line. You need to add penstrep to it along with 10-15% of FBS. Generally I add 10% FBS and Penstrep to my medium ( Invitrogen).
If you grow cells under anaerobic conditions for prolonged time or for some hypoxic assays, HEPES buffer is used. The glucose is the medium is converted to lactic acid rather than carbon dioxide and water. HEPES buffer will neutralize this effect.
Thank you Koula,
Concerning the FBS, is it heat inactivated or "plain"?
When should the cells start growing "normally"? I mean, after thawing they might feel not very good.. At what moment should I concider they're ok?
And, btw for the subculture - how often should it be done? Can I use trypsin? (I read the official guide - however very interested in "real" comments)
Thank you!
The FBS should always be inactivated and possibly filtered before freezing/using.
In general, cells start behaving "normally" 1 or 2 weeks after thawing. Your cells seems to be grow quite slowly, so it could take longer than that. You can tell they are ok when they grow at about the expected rate and you need to split according to recommendations.
I have no direct experience with these cells, so I will leave the other questions to people with more knowledge. But I would follow the guidelines, at least initially. Since they do not recommend trypsin, I would not use it. Also, you split them (subculture) when they are confluent or nearly so, I think this is valid for all cell types.
Best.
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