Fig. 3). RL
cells were seeded onto 384-well EPIC PF-04691502 A66 PI3K Abexinostat plates precoated with
or without having 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. Addition
of anti-IgM elicited a good shift in response that corre-
sponded to an improved mass inside of the sensing volume in wells coated with ICAM-1 (Fig. 3A). The peak response
was about 25 min publish anti-IgM application, fol-
lowed by a slow decay (Fig. 3A). The slow decay and
decreased mass inside the sensing volume would suggest
the achievable release of RL cells in the ICAM-1-coated
surface. From a practical viewpoint, this would be con-
sistent with immune cell extravasion and endothelial migra-
Indeed, the erythromyeloblastoid leukemia cell line,
K562, is reported to display dynamic LFA-1/ICAM-1 adhe-
sion whereby a time-dependent decrease in adhesion
strengthening that facilitates extravasion and transmigra-
tion was observed.
Importantly, RL cells did not seem to
associate together with the EPIC plate in the absence of ICAM-1,
supporting PF-04691502 A66 PI3K Abexinostat the notion the adhesion was LFA-1/ICAM-1
unique (Fig. 3A).
Pharmacological Characterization of your LFA-1/
ICAM-1 AssociationTo better comprehend the parameters from the 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 on the 25�C35 min time
interval, at which maximal peak response was recorded.
more 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 shown
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 in comparison with other blood-
Similarly, BIRT 377 is reported to
selectively inhibit LFA-1/ICAM-1 binding events in vitro
and in vivo.
Importantly, within the recent experiments, the two
BMS 587101 and BIRT 377 potently inhibited anti-IgM-
mediated LFA-1/ICAM adhesion PF-04691502 A66 PI3K Abexinostat with IC50
s of 23 nM and
332 nM, respectively (Fig. 3C). In contrast, BMS 587101
and BIRT 377 did not inhibit anti-IgM-mediated Ca
inside the FLIPR assay in both the Ramos or RL cells (Fig.
and Table 1). These data assistance the application from 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 of the instrument com-pounds validated in the FLIPR platform (Suppl. Fig. 2). In
common, the potency of the compounds was constant
within the RL cell line, irrespective of assay platform.
RN-486 and dasatinib had been most potent at inhibiting LFA-1/
ICAM adhesion (Fig. 4A).