In my experience working with primary cultures of mouse T-cells, it is very important. Normal bicarbonate buffer is CO2 dependent, which means if you are spending a lot of time working with the cells outside of the incubator it can change the pH (this is why the phenol red in the media changes color over time if you just leave the media out in room air versus in the incubator). HEPES is CO2 independent, and as such does a wonderful job buffering in an array of conditions. I think the important thing to remember is that HEPES should be added in addition to bicarbonate, not instead of bicarbonate. Bicarbonate has many metabolic functions as well as buffering capacities.

One important question to ask is what type of primary cells are you culturing? Some primary cells (fibroblasts) are very easy to culture and do not require many supplements to media, while others (t-cells for example) are very picky and require highly buffered and supplemented conditions.

I am unsure of any major differences between human and mice, as most people use a 10mM-25mM final working concentration of HEPES in their media regardless of species of origin.

I have attached a old, but gold-standard paper about tissue culture buffers. Hopefully it will help.

Good luck!

~Adam

In my experience working with primary cultures of mouse T-cells, it is very important. Normal bicarbonate buffer is CO2 dependent, which means if you are spending a lot of time working with the cells outside of the incubator it can change the pH (this is why the phenol red in the media changes color over time if you just leave the media out in room air versus in the incubator). HEPES is CO2 independent, and as such does a wonderful job buffering in an array of conditions. I think the important thing to remember is that HEPES should be added in addition to bicarbonate, not instead of bicarbonate. Bicarbonate has many metabolic functions as well as buffering capacities.

One important question to ask is what type of primary cells are you culturing? Some primary cells (fibroblasts) are very easy to culture and do not require many supplements to media, while others (t-cells for example) are very picky and require highly buffered and supplemented conditions.

I am unsure of any major differences between human and mice, as most people use a 10mM-25mM final working concentration of HEPES in their media regardless of species of origin.

I have attached a old, but gold-standard paper about tissue culture buffers. Hopefully it will help.

Good luck!

~Adam

Great answer,

Thanks a lot

Hi,

From my experience in skeletal muscle cell, nerve cell and lung epithelia cells as primary cultures, I only used Hepes to maintain cells under the microscope for 30minutes to one hour . Otherwise, I cultivated these primary cells without Hepes.

I test proliferation of normal or tumoral primary cells, with or without inhibitors in medium without hepes.

Best regards

Fab

Dependent on how long are you going to perform the expreriments on live cells outside of the incubator and which approach are you going to use for proliferation assay.

If you are planning to perform an experiment of 6hr and before then Hepes buffer alone would be fine. but for more than 6 hr experiment you should use appropriate media containing all the suppliments with HEPES. Rest It is totally depend on your experimental design.

I agree with previous answer. For my experiment added HEPES will use to cultivate for long duration (more than 6 days).

HEPES has the highest buffering capacity. That's the way to kep the culture conditions as long as possible constant (pH!!!)

You might want to look into adding b-mercaptoethanol, it is widely used in mouse lymphocyte cultures to enhance proliferation.

the reason of hepes is to keep the pH stable without a CO2 component,

We culture human and mouse PBMCs without HEPES and it works good.

But be carefull with its use. For some cells HEPES in the presence of fluorescent lights can be toxic. http://atcc.custhelp.com/app/answers/detail/a_id/84/~/hepes