associate with all the EPIC plate during the absence of ICAM-1,
supporting the notion the adhesion was LFA-1/ICAM-1
particular (Fig. Alisertib Stattic Entinostat 3A).
Pharmacological Characterization with the LFA-1/
ICAM-1 AssociationTo much better understand the parameters of your EPIC assay, we
titrated the anti-IgM-dependent response. The anti-IgM
response was dose dependent with an apparent EC50
��g/mL (Fig. 3B). Information have been taken at the 25�C35 min time
interval, at which maximal peak response was recorded. To
even further validate the platform, we examined the pharmacol-
ogy of two well-characterized LFA-1/ICAM-1 inhibitors,
BMS 587101 and BIRT 377. BMS 587101 has been proven
to inhibit LFA-1-mediated adhesion of T cells to endothelial
cells with an IC50
of twenty nM.
Moreover, BMS 587101 is
reported to be selective to LFA-1 when compared with other blood-
Similarly, BIRT 377 is reported to
selectively inhibit LFA-1/ICAM-1 binding events in vitro
and in vivo.
Importantly, inside the present experiments, the two
BMS 587101 and BIRT 377 potently Alisertib Stattic Entinostat inhibited anti-IgM-
mediated LFA-1/ICAM adhesion with IC50
s of 23 nM and
332 nM, respectively (Fig. 3C). In contrast, BMS 587101
and BIRT 377 didn't inhibit anti-IgM-mediated Ca
in the FLIPR assay in both the Ramos or RL cells (Fig. 2B
and Table 1).
These data assistance the application on the
EPIC platform for identifying inhibitors of LFA-1/ICAM
association in response to anti-IgM stimulation of RL cells.
We following examined the pharmacology in the instrument com-pounds validated while in the FLIPR platform (Suppl. Fig. 2). In
standard, the potency of your compounds was consistent
inside of the RL cell line, irrespective of assay platform.
RN-486 and dasatinib had been most potent at inhibiting LFA-1/
ICAM adhesion (Fig. 4A).
Similarly, these compounds
had been most potent at inhibiting anti-IgM-mediated calcium
flux while in the FLIPR assay (Table 1). The syk/BTK inhibitor
R406 displayed potency within the micromolar array in the two the
FLIPR and EPIC assays. The kind II inhibitor, compound 6,
displayed very little inhibition in the two cell-based assays. Also,
the covalent inhibitor, AVL-292, was an order of magnitude
more potent at inhibiting anti-IgM-mediated calcium flux in
Ramos cells when when compared with RL cells in either platform.
Figure 3. EPIC kinetic trace of RL cells stimulated with anti-IgM
(immunoglobulin M). (A) RL cells had been seeded on EPIC plates
coated with intercellular adhesion molecule 1 (ICAM-1; blue
trace) or uncoated (red trace). RL cells had been equilibrated for
approximately 2 h, followed by stimulation with anti-IgM. Within the
presence of ICAM-1, addition of anti-IgM improved the mass within
the sensing volume representing Alisertib Stattic Entinostat association of RL cells towards the EPIC
plate. This was absent in wells not coated with ICAM-1. Following
the steady-state transition, the mass gradually decreased, a response
that corresponds for the RL cells dissociating through the ICAM-1-
coated surface. N-DMR; unfavorable mass redistribution (decre