C646 SB203580 Nutlin

appreciable amounts of LFA-1 and that LFA-1 effector sys-
tems are downstream from Ca
flux, it had been not surprising
Figure 2. Pharmacological C646 SB203580 Nutlin inhibition of anti-IgM
(immunoglobulin M) mediated calcium flux in Ramos B cells. (A,
B) Ramos B cells were incubated with compound at various
concentrations before stimulation with anti-IgM. IC50
values for
the device compounds are reported in Table 1. The data from
representative experiments are shown as mean �� SD for each
concentration carried out in triplicate.that these inhibitors had no effect on Ca
flux (Fig. 2B and
Table 1).
In addition, both LFA inhibitors had no impact on
flux in RL cells, even further supporting that LFA-1/ICAM
association takes place downstream of Ca

From a routine-profiling viewpoint, the FLIPR-based
calcium flux platform yielded robust Z�� statistics depending on
DMSO versus CGI-1746 (10 ��M) handled cells. The typical
Z�� was 0.75��0.03, along with the Z�� assortment was 0.69�C0.79. The
signal�Cbackground (s:b) was 13.4��1.5, the s:b range was
Development of a Label-Free C646 SB203580 Nutlin Platform to
Measure B Cell Activation
As talked about, RL is often a human non-Hodgkin��s lymphoma B
cell line. RL cells express the integrin LFA-1, and associa-
tion with its ligand ICAM-1 mediates B cell adhesion. The
propensity for LFA-1 to associate with ICAM-1 is largely
dependent over the conversion of LFA-1 to an intermediate-
affinity conformation (Fig. 1).
The signaling cascades
elicited on BCR activation contribute on the conformational
shift demanded for LFA-1/ICAM-1 interactions.

The princi-ple of your EPIC platform is based on association of LFA-1
expressing RL cells to ICAM-1 coated around the EPIC plate
(Suppl. Fig. 3). We hypothesized that therapy of RL cells
with anti-IgM should shift LFA-1 expressed in RL cells to
an intermediate conformation capable of associating with
ICAM-1 rendered about the EPIC plate. Treatment method of RL cells
with inhibitors with the BCR signaling pathway must abro-
gate the LFA-1/ICAM-1 association (Suppl. Fig. 3). RL
cells were seeded onto 384-well EPIC plates precoated with
or without ICAM-1 and permitted to equilibrate for approxi-
mately 2 h in the EPIC. The equilibration time permitted the
cells to settle, resulting in a steady-state baseline.

of anti-IgM elicited a optimistic shift in response that corre-
sponded to an elevated mass within the sensing volume in wells coated with ICAM-1 (Fig. 3A). The peak response
was roughly 25 min submit anti-IgM application, fol-
lowed by a slow decay (Fig. 3A). The slow C646 SB203580 Nutlin decay and
decreased mass in the sensing volume would suggest
the achievable release of RL cells from your ICAM-1-coated
surface. From a functional point of view, this could be con-
sistent with immune cell extravasion and endothelial migra-
tion. Without a doubt, the erythromyeloblastoid leukemia cell line,
K562, is reported to display dynamic LFA-1/ICAM-1 adhe-
sion whereby a time-dependent lower in adhesion