Cells are grown at 20% oxygen which amplifies ROS production. This leads to oxidation of antioxidant pools. To avoid oxidative stress, most cultured cells amplify antioxidant defense however; certain cell types are unable to do so and thus beta-mercaptoethanol is added to aid in maintaining a reducing environment. For instance the insulinoma cell lines require beta-mercaptoethanol for its growth since the glutathione pool is highly oxidized. Suspension cell lines like PML cells and hybridomas also sometimes required added reducing agents for proper cell growth. Other reducing agents such as DTT can also be supplemented into the medium. However, one must exercise caution when adding these agents. Indeed, thiol agents like beta-mercapto and DTT can also autocatalyze ROS formation via the genesis of thiol radicals.

2-mercaptoethanol or beta mercaptoethanol is a reducing agent, thus when added to a cell line, it reduces toxic oxygen radicals.

I understand that much, but for what purpose would you use this? Not all cells require this, or it would be standard to add.

if you are culturing human cells, it is generally not necessary. it is recommended for mouse cells, but again, generally not many labs follow this protocol. however, there is a theory about using mercaptoethanol in culturing tumor cells, wherein it allows uptake of cysteine by creating a reducing atmosphere. (Bannai, 1992)

yes , you can use up to 50 Micro Molar concentration . Generally people use this in primary culture or where cell death is more.I used this and got positive result.

it seems that 2-ME is added MAINLY to mouse lymphocyte cultures, due to some empirical observations made by immunologists in the 1970s. It assists

in cysteine uptake Take a look at this link: https://lists.purdue.edu/pipermail/cytometry/2002-April/022078.html

Beta-mercaptoethanol (or alpha-thioglycerol) at a concentration of about 0.1 millimolar substantially enhances the plating efficiency of normal erythroid progenitor cells. It enhances the maximum number of colonies you can get with a saturating dose of erythropoietin, and it enhances the sensitivity to non-saturating doses of erythropoietin. "Old" (presumably oxidized) solutions of BME or ATG work as well as freshly prepared solutions. See Exp. Hemat. (1975) 3: 32-43 for details.

Most research groups use 2-mercaptoethanol or beta mercaptoethanol in stem cell and progenitor cell assays such as the erythroid colony assay mentioned by Fritz Sieber. We also use it for culture of a human preadiptocyte cell line derived from induced pluripotent stem cells that we are presently working with. It has a significant effect.

We use 0.05mM in RPMI to culture our THP-1s. Most folks seem to think it's a bit witchcrafty but the general consensus seems to be that it helps keep cysteine/GSH levels stable, maintain redox state. Others say it enhances antigen presenting and, as others have said above, may help limit clumping of the cells.

Cells are grown at 20% oxygen which amplifies ROS production. This leads to oxidation of antioxidant pools. To avoid oxidative stress, most cultured cells amplify antioxidant defense however; certain cell types are unable to do so and thus beta-mercaptoethanol is added to aid in maintaining a reducing environment. For instance the insulinoma cell lines require beta-mercaptoethanol for its growth since the glutathione pool is highly oxidized. Suspension cell lines like PML cells and hybridomas also sometimes required added reducing agents for proper cell growth. Other reducing agents such as DTT can also be supplemented into the medium. However, one must exercise caution when adding these agents. Indeed, thiol agents like beta-mercapto and DTT can also autocatalyze ROS formation via the genesis of thiol radicals.

I only use it inmurine bone marrow derived cultures (primary) to prevent oxidation due to build up of reactive oxygen species as this can induce maturation of the dendritic cells and/or cell toxicity. It is not necessary for standard immortalized cell lines.

Yes. We use 2-ME (1-5 x 10(-5) M) for many years in experiments with mouse splenocytes, bone marrow, B-1 and B-2 cells to induce primary immune response to T-dependent and T-independent antigens. 2-ME improves cell viability and immune response. Some years ago I have used 2-ME also for the cultures of human B lymphocytes. Good luck!

I have seen in PC 12 cells that 2-mercapto-ethanol enhances very rapidly intracellular glutathione level, and therefore protects the cells against mild oxidative shocks

I second here Ekaterina's comments. Several companies sell cell-culture grade ready-to-use 55 micromolar 2-betaME (1000x). I have used mostly for T cell in vitro cultures, as it prevents certain aminoacids from being degraded/metabolized/oxidized. it seems to be especially relevant for T cell survival/proliferation in the absence of other antigen presenting cells. Check http://www.ncbi.nlm.nih.gov/pubmed/11830651 for more details. I also guess it helps with oxidative stress, but you might want to be careful with this in case you are evaluating e.g ROS production or macrophage/neutrophil phagocytic activity or something similar. 

Hi!

do you add 2-ME to granulocytes culture separated from mice whole blood?

Hadas :)

Respected All, 

I am working on human myeloma cell lines RPMI 8226  we bought it from ATCC, and we  made cryos according to the protocol, but now when I revive those cryo. extensive clumps are being formed which could not be seen in the original stock supplied by ATCC. MY media constitutents are RPMI 1640, 4.5G/L glucose, 10mM HEPES, 2mM L-glutamine, 1mMsodium pyruvate, 1.5g/l sodium bicarbonate. 

plz give your suggestion as my thesis work is struck in between.

Shall I go for BME addition or what effect of lowering the glucose conc. or without HEPES. ??

Thanks and Regards

I always use 2-ME for my murine B cell culture RPMI -1640 media. this is required to protect from oxidative shock.

Has anyone used 2-mercaptoethanol in plant cultures?

In respect to the medium supplemented with mercaptoethanol, it can be stored (4ºC) for how long?

Thank you

In me experience medium supplemented with 2 mercaptoethanol can not be kept for more than 24h even at 4°C, it should be made fresh every day to avoid oxidation. 

Sincerely your's

D Duval Ph. D.

Yes.  We use 2-mercaptoethanol in certain "sensitive" melanocyte cultures.  In our case it seems to remove something toxic from the fetal calf serum, so for us, once it has been added to the medium, the effect is reasonably stable (for a few weeks).  However I'd agree that the reducing activity of ME won't last long in dilute solutions.

We use 100 uM, which seems to be harmless to cells generally (though a bit smelly... 1M stock prepared in fume hood).  We make a 1M stock with sterile distilled water, which can be stored at room temperature for a couple of months; and 10 mM or 20 mM stocks in sterile PBS, which can be stored in the fridge for about 3 weeks.  Have as little as possible air space in the container.   More-dilute solutions soon lose activity by oxidation by air.  

BTW, do not attempt to filter-sterilize pure ME (like DMSO) as it will dissolve materials out of the filter.  (Also out of rubber, so don't use rubber-lined caps). You can consider it already sterile - nothing will survive in this liquid.  (Again like DMSO).

Dear Dorothy,

Thank you soo much for your attention, this will help me a lot.

Just one more question please. For 10mM or 20mM stocks, do I need 1x or 10x sterile PBS ?

Best regards

1x.  You would be adding 1 part in 100 or 200 of this directly to your medium.

Unless you are adding it fresh daily, it is useless.

Another example: Yes, I use it for mouse and human induced pluripotent stem cell reprogramming or culture.

Hi. I have been struggling to grow Rin-m5F cell lines for some months now. they would attach and suddenly start to detach one by one till the flask is empty. Could it be the stress or what? I wasn't adding any 2-BM. I am using Gibco RPMI-1640 from Life etch and they saying it has NA bicarbonate but its not written like any other RPMI from Sigma indicated on the bottle. Please help.

regards Ananias

i have bmercaptoethanol from sigma with 14.3 M concentration.....what volume i have to add to prepare 10 ml of media from the pure liquid????

Hi everyone,

Very interesting post. I hope you can halp me even if it's not a recent post.

In my lab/department 2-ME is forbidden and I am would like to use DTT instead for murine B cells culture (isolated from spleen). At which concentration should I use it? Same as for 2-ME (5uM)?

We always use 2-ME for murine Embryonic stem cells culture DMEM.HG media. this is required to protect from oxidative shock.

we usually used in our PC 12 cultures higher concentrations of either beta mercaptoethanol 10-100 µM or DTT which is not toxic up to 100 µM (30 µM would be a good working concentration)

Hi to everybodies, I shoud use 1-Thiol 4.5x10-4M in pancreatic cell differentiation protocol, but I haven't it. Can I use 2BM? And what concentration?

thanks

Learn a lot from this post!

I use it for THP-1 human monocytic cells at 50 uM. I make up only a few mL of 1000x stock at 50 mM in water because I read somewhere that salts cause it to degrade quickly. (I don't even need that much, just the minimal volume to pass through a syringe filter to sterilize.)

Even so, I found it's only good for 3-4 days at most; the cell growth slows down noticeably if I use BME solution that is > 4 days old.

THP-1 control of bacterial infection is also significantly worse without BME in the medium. (EDIT for clarification: I'm doing mycobacterial in vitro infection experiments. This comment is unrelated to contamination of routine cell culture.)

Has anyone tried TCEP? It is reported to be much more stable than b-ME or DTT and not smelly.

Would lower oxygen level to physioxic levels be preferable to using 2-ME?

Thank you very much all for the answer

Shi-Hsia Hwa Hi ! do you mean if we dont use BME, cells are susceptible for bacterial infection ?

Kondareddy Cherukula Sorry for the lack of clarification. I am studying Mycobacterium tuberculosis infection in vitro. I mean during infection experiments, not with regard to contamination during routine cell culture.

Shi-Hsia Hwa Thanks for the clarification. However many people are culturing THP-1 without BME in media and no problems seen