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Ramos-Blue Cells - NF-kB/AP-1 Reporter B lymphocytes
B lymphocytes are key players in the adaptative immune system but are also prominent in the innate immune response. Consistent with their dual role, they
express Toll-like receptors (TLRs) and other pattern recognition receptors (PRRs) that allow them to discriminate among a wide spectrum of pathogen-associated
molecules (PAMPs). Upon PRR stimulation by PAMPs, various signaling pathways are induced leading to the activation of transcription factors, such as NF-kB
and AP-1, and the subsequent production of inflammatory cytokines.
Ramos-Blue Cells
Ramos-Blue is a B lymphocyte cell line that stably expresses an NF-kB/
AP-1-inducible SEAP (secreted embryonic alkaline phosphatase) reporter
gene. Ramos-Blue cells derive from a human Burkitt’s lymphoma which
is negative for Epstein Barr virus. They have the characteristics of B
lymphocytes and are routinely used as a model of B lymphocytes and for
apoptosis studies. The Ramos-Blue cell line was isolated for its ability to
respond to CpG ODNs (TLR9 ligands).
Ramos-Blue cells are responsive to NF-kB inducers, such as TNF-a and
TLR agonists. When stimulated, they produce SEAP in the supernatant
that can be readily monitored using QUANTI-Blue . QUANTI-Blue is
a SEAP detection medium that turns blue in the presence of SEAP (see
page 14). Levels of SEAP can be determined qualitatively with the naked
eye or quantitatively using a spectrophotometer at 620-655 nm.
Ramos-Blue cells are resistant to Zeocin .
Ramos-Blue -defMyD
Ramos-Blue -defMyD are MyD88-deficient cells. Due to their deficiency
in MyD88 activity, these cells are unable to respond to the activation of
receptors which signaling is dependent on MyD88, such as TLR7 and
TLR9. However, they are still responsive to TLR3 which signals through
TRIF independently of MyD88 (Figure 2).
Quality Control
Ramos-Blue cells express all TLRs and NOD1 mRNAs as detected by
RT-PCR. However, activation of NF-kB/AP-1 was only observed following
stimulation with TLR2, TLR3, TLR7, TLR9 and NOD1 agonists (Figure 1).
Considering the concentration of agonists used to stimulate these cells,
TLR3, TLR7, TLR9 and NOD1 responses are considered to be strong.
TLR2 response is detectable only at high concentrations of the ligands.
Responses to TLR4, TLR5 and NOD2 agonists are not detectable.
MyD88 deficiency in Ramos-Blue -defMyD cells was confirmed by
qRT-PCR.
Contents
Ramos-Blue cells are grown in IMDM medium, 2 mM L-glutamine, 10%
FBS supplemented with 100 μg/ml Zeocin . Each vial contains 5-7 x 10 6
cells and is supplied with 10 mg Zeocin . Cells are shipped on dry ice.
The cells are guaranteed mycoplasma-free.
Related Products
Zeocin , page 18 TLR Ligands , pages 77-80
QUANTI-Blue page 14 NOD Ligands, page 80
www.invivogen.com/reporter-cells
Figure 1: NF-kB/AP-1 activation in Ramos-Blue cells induced by various activators.
Cells were incubated with 10 μg/ml each of Pam3CSK4 (TLR1/2), FSL-1 (TLR2/6), HKLM
(TLR2, 1.10 9 cells/ml), poly(I:C) (TLR3), LPS-EK (TLR4), recFLA-ST (TLR5), imiquimod
and CL264 (TLR7), CL075 (TLR8), R848 (TLR7/8), ODN2006 and ODN2216 (TLR9),
TriDAP (NOD1), MDP (NOD2) and TNF-a.After 24h incubation, NF-kB/AP-1 activation
was assessed by measuring the levels of SEAP in the supernatant using QUANTI-Blue .
Figure 2: MyD88-dependent and -independent responses in Ramos-Blue and
Ramos-Blue -defMyD. Cells were incubated with 1 μg/ml poly(I:C) (TLR3), 10 μg/ml
CL264 (TLR7), 2 μg/ml ODN2006 (TLR9) and 100 ng/ml TNF-a. After 24h incubation,
NF-kB activation was determined using QUANTI-Blue .
PRODUCT QUANTITY CAT. CODE
Ramos-Blue Cells 5-7 x 10 6
cells rms-sp
Ramos-Blue-defMyD 5-7 x 10 6
cells rms-dmyd
69
INNATE IMMUNITY 3