Many thanks to all those who responded to my query re: use of beta
mercaptoethanol in cell culture.
Several folks asked that I post a summary of responses, so here goes:
********
You may have to look at some papers from the mid-late 1970s from Frank
Fitch, Howard Engers, J. Cerottini on the postive effects of 2-ME on
mixed
lymphocyte cultures and the proliferation/differentiation of
alloreactive
cytotoxic T lymphocytes (CTL). I was a grad student in Frank Fitch's
lab
and "why 2-ME" question used to come up periodically. There was never a
good explanation for how 2-ME worked, but it did something positive, so
we
always included it in T cell cultures.
Gib Otten
Chiron Corp
********
It will act as an antioxident to prevent free radicals. If your cells
are
sensitive it is essential for their growth and maintenence.
**********
Hi Bunny,
2-me is a reducing agent necessary to be added to help keep free radical
oxygen from affecting MOUSE cells. I capitalize the mouse because it is
generally unnecessary for human cells.
Randy T. Fischer
**********
Hi Bunny,
Many moons ago when I was (a) doing rat lymphocyte cultures and (b)
experimenting with Iscoves medium, I found it necessary to add about 50
micromolar 2-ME to the media, particularly for the rat cells. At that
time I believe the general opinion was that the 2-ME was acting as a
reducing agent and scavenging free radicals and other such nasties.
I just looked on Google for "mercaptoethanol"+"tissue
culture"+"rationale" and one of the results was the following, which
sounds more plausible:
http://216.239.37.100/search?q=cache:GWS5v70mPZEC:www.pharmanac.net/pdf_documentation/GIAF.pdf+mercaptoethanol+%22cell+culture%22+rationale&hl=en
(I couldn't connect to the source, so this is the cached version). It
says (exerpted):
Glutathione: Immunity:
The importance of glutathione and cysteine for lymphocyte functions
in vitro.
Studies of lymphocyte functions in cell cultures have been
greatly facilitated by the empirical finding that these
functions are strongly enhanced by thiol compounds
(Meister & Anderson, 1983; Ishii et al. 1987). This finding
is most strikingly exemplified by the fact that immuno-
logists have been adding 2-mercaptoethanol routinely to the
cell culture medium when studying immunological responses of murine
lymphocytes (Fanger et al. 1970).
Many years after these first experiments it became clear that
2-mercaptoethanol enhances the cysteine supply to the
lymphoid cells (Ishii et al. 1987), and thereby increases the
intracellular level of the cysteine-containing tripeptide
glutathione (see Fig. 1; Meister, 1983; Meister & Anderson,
1983).
<--- snip--->
Fanger MW, Hart DA, Wells JV & Nisonoff A (1970) Enhancement by reducing
agents of the transformation of human and rabbit peripheral lymphocytes.
Journal of Immunology 105, 1043-1045.
Ishii T, Sugita Y & Bannai S (1987) Regulation of glutathione levels in
mouse spleen lymphocytes by transport of cysteine. Journal of Cellular
Physiology 133, 330-336.
Meister A (1983) Selective modification of glutathione metabolism.
Science 220, 471-477.
Meister A & Anderson ME (1983) Glutathione. Annual Review of
Biochemistry 52, 711-760.
Hope this is some help!
All the best
- David
*****************
mouse cells, not human, require b-ME. If you do a proliferation assay
and
forget the b-ME (or grow HT-2 cells w/o b-ME) the results will speak for
themselves as to why to add it.
Lisa Geiselhart
*****************
Bunny,
Long ago (like 1979, when I was at the Clinic!), we used 2-ME as a
supplement in MEM or RPMI for lymphocyte activation studies. The info
at
the time was that cells would remain viable longer with the reducing
agent
in the media. Eventually, we stopped using it (don't remember precisely
why; probably we just forgot to add it one time) and the assays
functioned
well. The original Mishell-Dutton or Marbrook lymphocyte stimulation
assays
all included it, but I don't remember a specific function. Over the
years,
I have found that antigen and mitogen stimulation works fine in RPMI
without
the 2-ME, and the lab smells better! Carol DeLaMotte in surgical
research
at the Clinic, has a great deal of tissue culture experience and may
have
some insight into this.
Tom
Thomas S. Alexander, Ph.D. D(ABMLI)
****************
Hi Bunny
>From what I know 2-ME is added to mouse lymphocyte cultures to assist
in
cysteine uptake. Here are some references eluding to that. I am sure
there
are papers published prior to these. My apologies if this is more
information than what you wanted.
Roli
1: Int J Immunopharmacol 1992 Oct;14(7):1221-34
Effects of 2-mercaptoethanol and buthionine sulfoximine on cystine
metabolism by and proliferation of mitogen-stimulated human and mouse
lymphocytes.
Messina JP, Lawrence DA.
Department of Microbiology and Immunology, Albany Medical College, NY
12208.
Cysteine is an essential amino acid for lymphocytes and its anabolic
products are intimately involved in lymphocyte activation. The purpose
of
this study was to assess the uptake and subsequent utilization of
cyst(e)ine
by mitogen stimulated human peripheral blood mononuclear cells (PBMC),
to
evaluate the effect of an exogenous thiol, 2-mercaptoethanol (2ME), on
these
processes, and to compare human and mouse lymphocyte reactivities.
Unlike
mouse lymphocytes, the proliferation of human T-cells was inhibited by
addition of 2ME although 2ME enhanced cystine uptake. Optimal responses
to
T-cell mitogens (Con A and PHA) were obtained with a cystine
concentration
of greater than or equal to 25 and 200 microM for human and mouse cells,
respectively, and 2ME enhanced DNA synthesis of Con A-stimulated mouse
cells
regardless of the cystine dose; however, 2ME enhanced the response of
human
cells only in the presence of suboptimal doses of cystine. To assess
whether
2ME's inability to enhance human PBMC responses was related to their
glutathione (GSH) content, the human PBMC were pretreated with
buthionine
sulfoximine (BSO, an inhibitor of GSH synthesis). Even when the initial
intracellular GSH concentration was lowered to below that of mouse
lymphocytes, 2ME still inhibited proliferation. In contrast, addition of
2ME
to human PBMC maintained in the presence of BSO enhanced the
proliferative
response suggesting that a critical level of thiols is needed for
proliferation. The ability of 2ME to enhance proliferative responses in
cystine deficient medium supports this contention. Consistent with thiol
involvement in activation, Con A increased [35S]cystine uptake 2-fold
within
4 h of incubation and enhanced subsequent conversion of cystine into
cysteine and GSH. Interestingly, BSO treatment only slightly inhibited
Con
A-induced protein synthesis (5%), but it significantly suppressed
conversion
of cystine into cysteine or GSH (80-95%) and blocked DNA synthesis
(90%).
Overall, the results indicate that various differential thiol
characteristics must exist between human and mouse lymphocytes and that
a
reducing equivalent is necessary for DNA synthesis but not lymphocyte
activation.
2: Am J Med 1991 Sep 30;91(3C):140S-144S Related Articles, Books,
LinkOut
Modulation of lymphocyte functions and immune responses by cysteine and
cysteine derivatives.
Droge W, Eck HP, Gmunder H, Mihm S.
Division of Immunochemistry, Deutsches Krebsforschungszentrum,
Heidelberg,
F.R.G.
Mitogenically stimulated human peripheral blood lymphocytes and T cell
clones were found to have weak membrane transport activity for the
disulfide
cystine but strong membrane transport activity for the thiol amino acid
cysteine. Cysteine, however, is represented at the lowest concentration
among all protein-forming amino acids in the blood plasma. Complementary
laboratory experiments have shown that the cysteine supply is indeed
limiting for important lymphocyte functions. Proliferative responses of
mitogenically stimulated lymphocytes and T-cell clones and the
activation of
cytotoxic T cells in allogeneic mixed lymphocyte cultures are strongly
influenced by small variations in the extracellular cysteine
concentration
even in the presence of relatively high and approximately physiologic
concentrations of cystine. Cysteine can be substituted by
N-acetylcysteine
but not by cystine. The more detailed analysis revealed that the
extracellular supply of cysteine influences strongly the intracellular
level
of glutathione (GSH) and also the activity of the transcription factor
NF
kappa B that regulates the expression of several immunologically
relevant
genes. In vitro experiments including double-chamber experiments with
macrophages and lymphocytes revealed, moreover, that cysteine plays an
important role as a regulatory mediator between these cell types. The
cysteine supply is impaired directly or indirectly in several pathologic
conditions that are associated with immunodeficiencies, including the
acquired immune deficiency syndrome (AIDS). Cysteine or cysteine
derivatives
may therefore be considered for the treatment of patients with HIV-1
infection.
3: J Cell Physiol 1989 Oct;141(1):40-5
Involvement and relative importance of at least two distinct mechanisms
in
the effects of 2-mercaptoethanol on murine lymphocytes in culture.
Pruett SB, Obiri N, Kiel JL.
Department of Biological Sciences, Mississippi State University,
Mississippi
State 39762.
2-Mercaptoethanol (2-ME) exerts several effects on murine lymphocytes
in
culture that might explain its ability to enhance survival and growth of
these cells. The uptake of the essential amino acid cystine and
consequently
the maintenance of intracellular glutathione levels are enhanced by
2-ME.
Furthermore, 2-ME (even in the disulfide form) causes lymphocytes to
release
thiols into the culture medium. These effects might protect the cells
from
oxidative damage. The additional cystine provided by treatment of
lymphocyte
cultures with 2-ME might also allow adequate protein synthesis to
support
survival and/or growth. This study was conducted to assess the relative
importance of the antioxidant and protein synthesis effects of 2-ME. As
expected, 2-ME increased cystine uptake at all concentrations that
enhanced
growth and survival, but four nonthiol antioxidants that enhanced growth
and/or survival either did not substantially affect cystine uptake or
decreased it and did not affect the release of cystine or its products.
The
results presented here demonstrate that antioxidant protection is
necessary
and sufficient for lymphocyte survival and that cystine uptake in
untreated
lymphocytes is sufficient to support the protein synthesis needed for
survival and limited growth. However, we also noted that concentrations
of
2-ME that stimulated maximal growth more than doubled protein synthesis
as
measured at 8 hr. Thus the portion of the effects of 2-ME not accounted
for
by antioxidant action could be accounted for by enhanced protein
synthesis.
4: Cell Struct Funct 1989 Jun;14(3):287-97
Full replacement of 2-mercaptoethanol by cysteine plus selenium
compounds
in augmenting DNA synthesis of mitogen-stimulated mouse spleen
lymphocytes.
Ishii T, Sugita Y, Bannai S.
Institute of Basic Medical Sciences, University of Tsukuba,
Ibaraki-ken,
Japan.
Mouse spleen lymphocytes require 2-mercaptoethanol for maximal
mitogenic
activation in vitro. Previous studies indicate that the lymphocytes are
defective in the cystine transport activity and that they require
2-mercaptoethanol to utilize cystine. 2-Mercaptoethanol catalytically
carries cysteine moiety into the cells in a mixed disulfide form.
Because
cysteine is easily oxidized to cysteine in the culture medium, it has
been
not easy to precisely examine the effect of near-physiological
concentrations of cysteine on the activation of lymphocytes. By
controlling
the cysteine content in the medium, we have reviewed the effect of
cysteine
to see if cysteine replaces 2-mercaptoethanol in enhancing the DNA
synthesis
of lipopolysaccharide-stimulated lymphocytes. It was found that cysteine
was
less effective than 2-mercaptoethanol, and that cysteine fully replaced
2-mercaptoethanol when a selenium compound was supplemented. The effects
of
cysteine and selenium compounds were apparently independent and
additive.
Among the selenium compounds examined, sodium selenite and
L-selenocystine
were much more effective in stimulating DNA synthesis than sodium
selenate
and L-selenomethionine.
5: J Immunol 1983 Jan;130(1):362-4
Changes in intracellular glutathione levels in stimulated and
unstimulated
lymphocytes in the presence of 2-mercaptoethanol or cysteine.
Zmuda J, Friedenson B.
Certain low m.w. thiol compounds can enhance various in vitro lymphocyte
mitogenic responses, whereas reagents that react with sulfhydryl groups
are
potent cell poisons. The possible targets for thiols and thiol reagents
could include membrane and cytoplasmic proteins, some of which may
regulate
lymphocyte proliferation. As a step toward understanding how lymphocyte
proliferation is enhanced in the presence of thiols, we studied the
appearance of both protein and non-protein sulfhydryl groups in
stimulated
lymphocytes. To help clarify the role of the cellular targets for
thiols, we
studied the time course of appearance of both protein and non-protein
sulfhydryl groups as lymphocytes proliferate under various conditions.
In
the presence of an appropriate concentration of the thiol
2-mercaptoethanol
(2-ME), there is a substantial rise in the level of intracellular
glutathione for lymphocytes stimulated with the mitogen concanavalin A
(Con
A). Otherwise, the level of intracellular glutathione declines, even for
Con
A-stimulated cells, but the presence of 2-ME partially prevents this
decline. Increasing the amount of cysteine in the medium to a level that
enhances cell proliferation leads to effects similar to those obtained
with
2-ME. Thus, apparently one effect of various thiol reducing agents is to
enhance the production of glutathione in lymphocyte mitogenesis and to
protect against the loss of glutathione that occurs in resting or
proliferating lymphocytes.
*************
--
Bunny Cotleur, M.S.
Sr. Technologist
Cleveland Clinic Foundation
Neurosciences NC30
9500 Euclid Avenue
Cleveland, OH 44195
cotleur@ccf.org
(216) 444-1164
fax (216) 444-7197
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