400 mOsmol is definitely too high. It will cause osmosis of water out of the cells, resulting in shrinkage. Anything over 330 or so is too high.

Why are you adding bicarbonate and HEPES to DMEM? DMEM is already buffed and includes bicarbonate and phosphate.

You're adding full 2nd helping of bicarbonate and then HEPES on top of that?

HEPES is normally used as replacement buffering system when not using CO2.

What exactly are you trying to achieve? Are you making your own DMEM or using a commercial formulation?

Anyhow, just adding serum, pen/strep, glut and pyruvate to normal commercial DMEM should be no problem. In a standard CO2 incubator 

If you are using commercial DMEM, you should NOT be adding anything extra, other than your serum and perhaps your antibiotic. Check the manufacturer's product information for how much salts their DMEM contains. 

Adding a high concentration of salts into your culture medium will cause your cells to shrink and die. 

I have never chacked the osmolality of the culture medium I used but 407 mOsm seems a liitle bit too high (~350 may be more close to physiological level). Did you add  2mM Glutamine, Na-bicarbonate (3.7g/L), sodium pyruvate and HEPES yourself? Any reason why you add two more buffer into DMEM? Or, you actually measured the osm after culture? Even we put humidity in the incubator the osmilality can literally increase 10-15% after 48 hours with the lid of the plate on.

I'm culturing primary hippocampal neurons in Neurobasal medium, which has an osmolarity  235 mOsm. I did cell stress experiments with my cells to induce the heat shock response by increasing the osmolarity. They tolerated up to 435 mOsm quite well (for up to 72h) but died at higher osmolarity. But these were cells already cultured for 14 days in the normal osmolarity medium. So I would guess that you should try another medium.

The osmolarity of DMEM is listed commercially as 322-374 mOsm, so (from what Patrick says), everyone's cells grown in DMEM are spending a lot of energy on it.  Hardy cells like cancer cells are ok like that.  But (Vini), it sounds as if you are adding extra bicarbonate etc?  3.7g/L bicarbonate is 44 mM, more than 44 mOsm after dissociation.  Also this would make the pH very unfavourable.  DMEM is designed for use with 10% CO2 (I can tell you that having  trained with Dulbecco).  With no extra buffers and 10% CO2 it will give pH 7.4 (orange-red).  With 5% CO2 it gives pH 7.7 (pink) which is quite toxic for some cells, so if you add even more bicarbonate you may be killing the cells with high pH as well as high osmolarity.

There is nothing inherently wrong with using a combination of HEPES and Bicarbonate buffering. Indeed this is an excellent way of keeping the pH stable within your cultures when they are outside the 5% CO2 environment of an incubator. Reliance on CO2/ Bicarbonate alone can lead to pH rising quite significantly when cultures are outside the incubator, especially if the medium is shallow or exposure to normal air prolonged. Having said that, it is vital that you make sure additions do not cause hyper-osmolarity. 3.7g/l NaHCO3 is probably higher than you need, especially with HEPES present too. Around 2.3g/l should be sufficient. Take care too with any adjustments with acid / alkali additions to balance the pH of buffer stocks or media. Yo-yo-ing around the desired pH will bump up the osmolarity, (often hidden if someone else has performed this). Small changes like this should reduce the volume of any water you need to add to restore medium osmolarity to normal.

I like the point that Patrick Duggan made re: energy expenditure. You are after far more than cell survival. Correct osmolarity and pH are vital in avoiding abnormal expenditure of energy on homeostasis. The latter will lead to changes in metabolism that are difficult to predict and results that are harder to reproduce and interpret.

PS to my previous comment - standard DMEM already contains 44 mM bicarbonate, for use with 10% CO2, whereas media designed for use with 5% CO2 like RPMI, MEM and McCoy's tend to contain around 24-26 mM bicarbonate (2.2-2.4 g/L, as Jim C just mentioned).

There is no point in adjusting the pH of your medium with strong acid or alkali before putting in the incubator, as this will be swamped by the buffering power of the CO2-bicarbonate system, and also altered by warming to 37C.  If you want to achieve a specific pH in your incubator, you can make a series of bicarbonate (and/or HEPES) concentrations in screw-topped tubes, leave them in the incubator overnight, tighten the tops, transfer to a 37C waterbath, and measure each pH quickly after opening.

I used earlier early embryo culture medium by adjusting to 281-303 mOsmol/kg. I do not not if it changes a bit once it gets equilibrated with CO2

Patrick - actually MEM (Eagle's minimal essential medium) was designed to be isotonic, and it is, roughly.  DMEM (Dulbecco's modification of Eagle's medium) apparently arose when Marguerite Vogt wanted her transformed cells to be able to grow over the weekend.  Starting with MEM, she doubled the concentrations of most amino acids and glutamine, and also of bicarbonate and CO2 to increase buffering and stop the cells acidifying the medium in 3 days.  Of these, the bicarbonate-CO2 system has much the biggest effect on osmolarity.

Thanks everyone for your very useful comments and suggestions. Although the medium I used earlier for cell culture was DMEM plain (without sodium bicarbonate), the DMEM I have been using for B50 cells is a different one, and upon rechecking, it turns out that it already has 44mM of bicarbonate. So I was adding the amount again! I will make the formulations again - thanks everyone!

@ Dorothy, Jim, Patrick - All the DMEM data sheets say the following "DMEM uses a sodium bicarbonate buffer system (3.7 g/L), and therefore requires a 5–10% CO2 environment to maintain physiological pH." This was the reason I was adding 3.7 g/L of NaHCO3. Now if I am using DMEM plain and that if my incubator is the one with 5% CO2, do you suggest I should use 2.2-2.4 g/L bicarbonate only? And also, should I continue to use HEPES or its redundant?

About the shrinkage of cells: I am not sure if my cells shrink, but they certainly dont die. I have cultured them on coverslips for up to 12 days, and they survive. I must now check their morphology after culturing them in the isotonic medium.

Hi Vini,

Yes, you should use the lower level of bicarbonate if your incubator has 5% CO2.  I believe the manufacturers' data sheets fudge the issue, perhaps to try not to offend those users who are in the habit of using standard DMEM with 5% CO2.  However this does definitely give a high, non-physiological pH of 7.7 or higher (in various media) with 5% CO2.  You can tell by the phenol red colour if nothing else - pH 7.4 in medium without serum is a bright red with no trace of pink. 

I don't have any data for the effect of HEPES; this will indeed make the pH more stable when the cultures are taken out of the incubator, but it will also change the equilibrium pH at 5% CO2.  If you want to use HEPES, you could do a bicarbonate-pH curve dose-response for the DMEM + HEPES, in the way I described in a previous answer.

Hope that helps!

I would recommend that you keep the HEPES in to combat pH changes likely to occur outside the CO2 incubator environment, especially if media is shallow or you are actually collecting data after any variable periods of exposure to normal air. I'm told that there are a few cell types sensitive to HEPES, (I've forgotten which), but apart from those very rare reports I don't think there is a downside to using HEPES. I'm biased as I found it so useful for my own work which covered everything from microtitre to 10 stack cultures, and therefore needed media robust enough to cover wide circumstance.  One thing is for sure, you don't want to have two versions of each media type you use, (i.e. with / without HEPES). I would follow Dorothy's previous answer to identify the best combination of NaHCO3 and HEPES to get the best pH for the medium at 37 deg C. Good luck.

Thanks!!

Human Blood Osmolality in a non-diseased person is 275-295 mOsm/kg but I don't know if this info has any connection to your doubt

Reference: Jacques Wallach: 'The interpretation of diagnostic tests', 5th edition, 1992.

ISBN 0-316-92050-9

Does anyone know if changing FBS (0.5% to 10% range) concentration changes osmolarity of standard RPMI based media?

@Amanda Balaban No idea of lab work, I'm a clinician, but for what I remember about the physical chemistry I was taught 50 years ago, I'd say yes, FBS -what is this? concentration changes could probably change osmolarity. Can it? Salut +

Dear Joe Graymer , ....if i guess hopefully right, then

".. FBS -what is this? "

should be read as: "fetal / foetal bovine serum " (cf. also: https://acronyms.thefreedictionary.com/FBS first place result but always a source for other "unknown" abbreviations.

Dear Amanda Balaban ...(apologies for lengthiness)

Regarding increasing the FBS concentration ("...changing of from 0.5 to 10% range...") in general increasing medium osmolarity above - say- 400 to 450 mosmol may(should) result in damage or further on even necrosis of cells in culture, e.g. see:

(featuring Osmolalility and Osmolarity issues of cell culture media):

https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/osmolality , find

"F. Feyerabend, in Biomaterials for Bone Regeneration, 2014", (Osmolality-osmolarity), bottom sentences:

Quote: "...At values below 0.4 Osmol/kg cells will survive the treatment with extracts (the osmolality of cell-culture medium with foetal bovine serum (FBS) lies in the range of 0.33–0.34 Osmol/kg), but higher values will lead to cell necrosis due to osmotic shock (Fischer et al., 2011)." End of Quote.

Increased osmolarities usually are achieved by addition of ions (and adjusted by such like potassium chloride and sodium chloride).

"....Osmolality of FBS, a measure of the concentration of solutes like salts and sugars, should be similar to culture media to avoid osmotic shock that may impact cell viability. "(quote found at:

"Understand the Basics of Fetal Bovine Serum (FBS)" , cf. https://www.thermofisher.com/at/en/home/references/gibco-cell-culture-basics/cell-culture-environment/culture-media/fbs-basics.html .

"FBS: Osmolality (280–340 mOsm/kg H2O) ", cf.:

https://www.thermofisher.com/at/en/home/life-science/cell-culture/mammalian-cell-culture/fbs/value-fbs.html

FBS -osmolality - according to data sheets of producers (see also URLs below) - mostly (and dependant on manufacturer) is defined as measuring ~280 - 360 mOsm/Kg H2O [an other source: 0.33–0.34 Osmol/kg H2O] which means (if I am righ) that a 1 (one)% FBS-concentration in solution exerts only 2.8-3.6 mOsm/Kg and a 10% (ten) FBS-concentration therefore adds 28-36 mosmol/Kg H2O to the final osmolarity of the medium, which IMHO would not exceed certain 'integrity limits' of the "viability of cells" in a "physiological cell medium" [say: initially adjusted to 300 mosmol (+/-15) mosmol ] itself.

But it may depend also on the cell type you have to manage whether increased osmolarities are favourable for cells or not.

(e.g.: for effects of varied FBS-concentrations on osteocytes cf.:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2792074/ =

Villanueva et al, ‎2009, in: Tissue Eng Part A. 2009 Oct; 15(10): 3037–3048 =

RESEARCH GATE -

Article Medium Osmolarity and Pericellular Matrix Development Improv...

cf. Fig.4 A and 4B for the effect of (FB) serum concentration on chondrocyte survival.

[PDF (!NB!: in the following URL delete the two underline characters __ in between https and :// in your browser prior to linking....cf.

https__://www.ncbi.nlm.nih.gov/pmc/articles/PMC2792074/pdf/ten.tea.2009.0001.pdf ]

https://www.sciencedirect.com/topics/immunology-and-microbiology/osmolality

find also facts about FBS: : https://www.sciencedirect.com/topics/medicine-and-dentistry/fetal-bovine-serum,

You might check about osmolality/osmolarity values for FBS at URLs:

https://www.rmbio.com/fetal-bovine-serum

https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/p12203.pdf.

http://himedialabs.com/TD/RM9954.pdf

https://fcs-free.org/uploads/media/5964dd50c0821/FBS%203.pdf

Disclaimer:

No affiliation to or financial interest in any company mentioned in this answer.

Thanks for the recommendation, Dr. Korneev and greetings from the antipods to Australia !

Note added - because I forgot to mention mO regarding the RPMI issue:

What I should have added to my answer above was that at least 1 supplier of RPMI (there might be other suppliers too) (Disclaimer as above in my answer [020] applies too) in the MSDS / TechDataSheet does mentiongeneral physical data, including pH and osmolality:

cf:

"SIGMA RPMI-1640 MEDIUM With L-Glutamine and Without Sodium Bicarbonate HYBRI-MAX® Product Number R5382 Storage Temperature 2-8°C

„…

Osmolality* 237 mOsm/kg H2O ± 5% [without sodium bicarbonate] -

Osmolality* 279 mOsm/kg H2O ± 5% [with sodium bicarbonate]“

*pH and osmolality determined without adjustment to pH 4.0with 1N HCl.

[Source cf.: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/5/r5382dat.pdf ]

So you might be able (with regard to the addition of 0.5 % ==> 10% FBS, see the 'calculations' in Re [020] ) to determine the risk to get over / beyond or better: to exceed the usually recommended / known limit of "viability" and "integrity limit" of - roughly spoken - 350-450 mosmol.

About 20 years ago, we found, but not published, that about 10% increase in osmolality leads to activation of "volume-activated chloride channels" in HeLa cells.

Thus, 330 mOsM/kg and higher osmolality will affect ionic homeostasis, various ion (primary Ca2+)-dependent pathways, and cell development.

To reduce your media osmolality by 100 mOsM/kg you can either dilute your media with H2O (by adding about 100/407*100% = 24% of additional volume with water), or to reduce concentrations of your initial components (e.g., by decreasing concentration of Sodium Pyruvate by about 50 mM).

What you choose depends on what component concentrations are more important for you in the media.