HEPES in RPMI - is it necessary? - HEPES in RPMI - is it necessary? (Jan/07/2008 )

I am going to use RPMI without phenol red but it will also come without HEPES (from invitrogen). If I am using 5% CO2 for my culture do I need to add it or is it okay with out??

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If you dont grow cells to very high density you can use a medium without it. HEPES gives extra buffering capacity.

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Dear maria123,

The 2 main buffering systems in culture media are NaHCO3 and HEPES. If you use a CO2 Incubator AND OPEN vessels then you have to use NaHCO3 as a buffer. If you want to use a closed cell culture system, then you use HEPES Buffer, generally at a concentration of 25mM. This will buffer your media but is less efficient and is an old fashioned method. In the days before 0.22uM filter topped culture vessels, researchers used a closed system in order to prevent contamination, generally from the incubator environment. These days most flasks have these filters and NaHCO3 buffered systems rule.

The best thing is to look at the media constituents in the Gibco, PAA, Sigma, Flow catalogues, in order to check that Bicarbonate is there.

Hope this is useful

Kindset regards

Rhombus

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Thank you Rhombus for the information of the buffering systems.

I have learnt so much tissue culture technique from you.

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Hi Maria

We recently asked ourself the same question as we switched from HEPES-buffered to HEPES-free RPMI-1640 medium. This resulted in a temporary slower cell growth.

Therefore we tested both media on one of our fast growing cell lines. We grew the cells 10 days without splitting them (triplicates). Cells in both media grew similarly (exponentially) until day 3. In the HEPES-free medium cell number decreased after day 3., whereas cells grown in HEPES-buffered medium continued to grow exponentially until day 4-5 after which cell number declined.

Right now our technician is running a small experiment where she adds HEPES to the medium. The reason for this is that the two media are produced by different companies and we want to be sure that the HEPES-free medium is ok. I could send you more details if you are interested.

I think we will decide to go by the HEPES-buffered, even though the HEPES-free medium appears to be just as good as long as medium is changed and/or cells split every 3 days.

Hope this helps

Kirsten
Aalborg Hospital
Department of haematology
www.blodet.dk

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