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MEF Cells
C3H/TLR4mut & C3H/WT MEFs
TLR4 Reporter Murine Embryonic Fibroblasts
C3H/TLR4mut and C3H/WT MEF cell lines were isolated from C3H/HeJ
(TLR4-deficient) and C3H/HeN (wild-type) mouse embryos respectively.
They stably express an NF-kB-inducible SEAP reporter construct that
allows to monitor in a simple and convenient manner the activation of
NF-kB.
C3H/WT MEFs express high levels of TLR2 and TLR4 and low levels of
TLR3 and TLR5. The presence of TLR2, TLR3, TLR4, or TLR5 agonists
triggers a signaling cascade in C3H/WT MEFs leading to the activation of
NF-kB and the subsequent induction of SEAP. The amount of SEAP
secreted in the supernatant can be readily detected when using
QUANTI-Blue , a SEAP detection medium. In C3H/TLR4mut MEFs, TLR4
agonists do not induce the activation of NF-kB and the production of
SEAP in contrast to TLR2, TLR3 and TLR5 ligands. Thus these two cell lines
provide a useful tool to determine whether a given compound is a specific
TLR4 agonist. They can be used to assess the purity of a given LPS by
detecting the presence of contaminants that stimulate TLR2 (see graph,
LPS-EB standard versus LPS-EB ultrapure).
Contents
C3H/TLR4mut and C3H/WT MEF cell lines are grown in DMEM medium
with 2mM L-glutamine, 10% FBS supplemented with 10 μg/ml blasticidin.
Each vial contains 5-7 x 10 6 cells and is supplied with 1 mg blasticidin.
Cells are shipped on dry ice. The cells are guaranteed mycoplasma-free.
C57/WT MEFs
RLR Reporter Murine Embryonic Fibroblasts
MEFs produce IFN-b in response to viral infection in a RLR-dependent
manner1 . Thus, these cells are commonly used to study the RLR pathway.
C57/WT MEFs were isolated from embryos under C57BL/6 background
and immortalized with the SV40 large antigen. They stably express a SEAP
reporter gene inducible by NF-kB and IRF3/7 providing a convenient
method to monitor the activation of these transcription factors upon
stimulation with a RIG-I/MDA-5 ligand.
C57/WT MEFs express both RIG-I and MDA-5. Stimulation of C57/WT
cells with poly(I:C)/LyoVec complexes induces the secretion of SEAP in
a dose-dependent manner (see figure). In contrast, stimulation with naked
poly(I:C) has no effect on SEAP secretion although the cells express also
TLR3. These data confirm that C57/WT MEFs respond to viral dsRNA
primarily through the RLR pathway1 . Both RIG-I and MDA-5 appear to
respond to transfected poly(I:C) in MEFs2 .
1. Kato H. et al., 2005. Cell type-specific involvement of RIG-I in antiviral response.
Immunity 23:19-28. 2. Venkataraman T. et al., 2007. Loss of DExD/H box RNA helicase
LGP2 manifests disparate antiviral responses. J Immunol. 178:6444-6455.
Contents
C57/WT MEFs are grown in DMEM medium with 10% FBS, 2 mM
L-glutamine and supplemented with 100 μg/ml Zeocin and 3 μg/ml
blasticidin. Each vial contains 5-7 x 10 6 cells and is supplied with 10 mg
Zeocin and 1 mg blasticidin. Cells are shipped on dry ice. The cells are
guaranteed mycoplasma-free.
www.invivogen.com/reporter-cells
TLR stimulation profile of C3H/TLR4mut and C3H/WTMEF cells. Cells were
incubated with TLR agonists: Pam3CSK4, 100 ng/ml (TLR2), poly(I:C), 10 μg/ml
(TLR3), MPLAs, 10 μg/ml (TLR4), LPS-EB standard, 10 μg/ml (TLR4, TLR2), LPS-EB
ultrapure, 10 μg/ml (TLR4), LPS-RS, 10 μg/ml (TLR2), flagellin, 1 μg/ml (TLR5). After
24h incubation, TLR stimulation was assessed by measuring the levels of SEAP in
the supernatant by using QUANTI-Blue .
PRODUCT QUANTITY CAT. CODE
C3H/TLR4mut MEFs 5-7 x 10 6
cells mef-c3h4m
C3H/WT MEFs 5-7 x 10 6
cells mef-c3hwt
RLR stimulation in C57/WT MEFs. C57/MEFs were incubated with increasing
concentrations of poly(I:C) or poly(I:C)/LyoVec complexes prepared
extemporaneously at a 1:12 ratio. After 24h incubation, RLR stimulation was assessed
by measuring the levels of SEAP secreted in the supernatant by using QUANTI-
Blue , a SEAP detection medium.
PRODUCT QUANTITY CAT. CODE
C57/WT MEFs 5-7 x 10 6
cells mef-c57wt
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INNATE IMMUNITY 3