There is growing demand for more physiologically-relevant assay platforms, leading to increased adoption of both label-free technologies and stem cells in research. Automated systems are also needed to facilitate research by increasing cell-based assay efficiency, reproducibility, and performance. To address these needs, methods utilizing the PerkinElmer EnSpire™ Multimode Plate Reader with Corning® Epic® label-free technology and the JANUS® Automated Workstation have been developed to non-invasively identify and characterize multiple Ion Channel and GPCR activation in Lonza® CloneticsTM Human Umbilical Vein Endothelial Cells (HUVEC) and PoieticsTM human Mesenchymal Stem Cells (hMSC). By monitoring the ligand-induced dynamic mass redistribution (DMR) in living cells with no need for cellular or ligand modification, the EnSpire label-free platform detects cellular responses from endogenously expressed receptors and ion channels. This obviates the need to engineer cells to over-express receptors of interest, thus greatly reducing the possibility of altering cellular biology. These data demonstrate the applicability of label-free technology for primary and stem cell research and the utility of liquid handling automation to ensure highly reproducible data from these primary cell assays. For more information about the EnSpire™ Multimode Plate Reader, please visit http://bit.ly/17fwGwx For more information about the JANUS® Automated Workstation, please visit http://bit.ly/1cdJpSi

1. ®
Lonza
Non-Invasive Label-Free Studies of Receptor Activation in
Primary & Mesenchymal
® Multimode Plate Reader & JANUS® Automated Workstation
Stem Cells Using the EnSpire
Heidi Morgan, M.S., Vincent Dupriez, Ph.D., Tim Cloutier, Ph.D., PerkinElmer, Inc. Waltham, MA USA
Kristin Atze, Leon de Bruin, Lonza Cologne GmBH, Koeln, Germany
ATP M anual
Buffer M anual
20
0
20
25
HUVEC (3000 c/w)
40
Isoproterenol
Histamine
20
ATP
0
-12
-10
-20
-8
EC50
0.0002087
6.153e-010
Ligand
Rim
lon
e
µM
]
Response (pm)
25
1
0
-8
-50
-6
-4
[Ligand], M
-2
Bithionol
Pinacidil
-100
-150
Bithionol
Pinacidil
EC50
2.871e-005
9.240e-005
Label-free robustly monitors the activation of different ion channels in hMSCs.
• The absolute response (pm) +/- SD of mean with various ion channel activators and
several negative control compounds.
• Capsaicin (a known activator of the TRPV1 vanilloid/capsaicin receptor, potentially
activating another type of ion channel at the high concentration used here) and
Thapsigargin (potent inhibitor of sarco-endoplasmic reticulum Ca2+-ATPases),
provide large positive pm responses in hMSC.
• Bithionol (an activator of calcium-activated potassium channels) and Pinacidil (an
activator of ATP-sensitive potassium channels) provide large negative pm responses.
60
-8
-50
-6
Isoproterenol
Histamine
ATP
-4
[ATP], M
Three cell types were used to test the response of either recombinantly or
endogenously expressed Purinergic receptors to ATP: Human astrocytoma
cells (1321N1), human mesenchymal stem cells (hMSC), and human umbilical
vein endothelial cells (HUVEC). A robust 50-fold window (pm) is observed
with the endogenously expressed receptors, demonstrating the sensitivity of the
EnSpire with label-free platform.
80
2500 c/w
5000 c/w
10000 c/w
15000 c/w
40
20
0
-6
-4
[Ligand], M
EC50
9.031e-007
3.729e-007
6.034e-007
7.924e-007
0
-10
-8
-6
-4
-2
6
[Ligand], M
-20
EC50
~ 1.949e-009
2.146e-006
2.143e-006
Isoproterenol
Histamine
ATP
EC50
1.287e-009
2.024e-007
1.438e-006
Label-free ATP Receptor Antagonist Studies
in hMSC (2500 c/w)
100
3000 c/w
Response (pm)
60
ATP
20
-12
100
1500 c/w
300
Histamine
-2
Label-free Histamine Receptor Antagonist Studies
in hMSC (2500 c/w)
ATP Dose Responses Against
Titrating HUVEC Densities
ATP Dose Responses Against
Titrating hMSC Densities
40
Representative DRCs for several agonists in both cell types:
• Histamine and ATP act in a similar manner against the endogenous
Histamine and Purinergic receptors in both hMSC and HUVEC (positive
DMR response).
• Isoproterenol has a differential effect in hMSC relative to the HUVEC
system (negative DMR response).
• This points to potentially shared (ATP, Histamine) or divergent
(isoproterenol) couplings and/or divergent cellular response to a
common coupling for the receptors of these ligands.
6000 c/w
200
12000 c/w
100
0
-10
-2
-100
-8
-6
-4
-2
Response (pm)
132N1-rP2Y11 (10000 c/w)
hMSC (2500 c/w)
HUVEC (3000 c/w)
EC50
7.469e-007
7.923e-007
5.949e-007
-4
Clobenpropit
50
Histamine
0
-10
-8
-6
-4
Cell density titrations of hMSC or HUVEC vs. ATP performed to identify
the optimal assay signal and window for study.
• Lower numbers of cells per well increases the assay window by ~ 3-fold
relative to higher densities, thus providing an additional cost savings
benefit when running this simple assay workflow.
Suramin
0
-10
-8
-6
-4
-2
[Ligand], M
-50
-50
12000 c/w
6000 c/w
3000 c/w
1500 c/w
ATP
50
[Ligand], M
[Ligand], M
EC50
7.994e-007
4.168e-007
5.278e-007
7.876e-007
-2
Response (pm)
-10
-6
[Ligand], M
0
Response (pm)
Label-free Ligand Dose Response Curves
on HUVEC (12000 c/w)
Response (pm)
hM SC (2500 c/w)
50
Corning® and Epic® are registered trademarks of Corning Incorporated.
3-6 reps per bar
The label-free platform robustly monitors the activation of GPCRs
coupled to various pathways within multiple cell types.
• The absolute response (pm) +/- SD of mean with various GPCR
activators and several negative control compounds.
• AM251, LPI and rimonabant are three known agonists of the GPR55
cannabinoid receptor, which is known to couple only to Gα12/13 proteins.
Response (pm)
132N1-rP2Y11 (10000 c/w)
150
15000 c/w
10000 c/w
5000 c/w
2500 c/w
[Ligand], M
Isoproterenol
200
-20
0
Ligand
3-6 reps per bar
60
250
-8
AM
AM
25
1
Ligand
3-6 reps per bar
Label-free Ligand Dose Response Curves
on hMSC (2500 c/w)
ATP Effects On Different Cell Types
-10
-2
0
0
Robust Label-free Detection
-12
-4
50
Manual
0.71
6%
Bu
ffe
rA
EC50
8.941e-007
3.329e-006
-6
Label-Free Response with Ion Channel Ligands
in hMSC (2000 c/w)
µM
]
Automated
Z'
0.81
CV, max
3%
40
20
All liquid handling steps were performed both manually and on the
JANUS Automated Workstation with comparable results.
• The advantage of automating the label-free steps is to provide a robust and
reliable easy-to-use protocol resulting in significant time savings and
enhanced reproducibility.
3
20
Well Number
[Ligand], M
ATP (Automated)
ATP (Manual)
20
15
10
-8
Capsaicin
Thapsigargin
µM
]
5
0
60
µM
]
0
-2
Thapsigargin
20
-20
µM
]
-4
Capsaicin
-10
µM
]
-6
40
80
ne
[25
Ca
0
ps
ac
in
[25
0
Ca
ffe
ine
Ni
[25
flu
0
mi
cA
cid
Th
[25
ap
0
sig
arg
in
[40
Pip
eri
din
e[
25
Bi
0
thi
on
ol
[25
0
Pin
ac
idi
l [2
Iso
50
va
gu
cin
e[
25
Ga
0
bo
xa
do
l [2
50
-8
[16
µM
LP
]
I [7
on
0µ
ab
an
M]
t [2
Br
00
ad
µM
yk
ini
]
His
n[
tam
1µ
M]
ine
[50
0µ
PG
M]
E1
[25
0µ
PG
M]
E2
[50
µM
S1
]
P[
5µ
AT
M]
P[
Fo
50
rsk
0µ
oli
Iso
M]
n[
pro
50
ter
0µ
en
M]
ol
[50
0µ
M]
LP
A[
50
NE
µM
CA
]
[50
µM
Bu
]
ffe
rA
lon
e
-20
-10
[7
µM
LP
Rim
]
I [8
on
0µ
ab
M]
an
Br
t [9
ad
0µ
yk
ini
M]
n[
His
10
tam
0µ
M]
ine
[10
0µ
PG
M]
E1
[10
0µ
PG
M]
E2
[20
µM
S1
]
P[
2µ
AT
M]
P[
Fo
10
rsk
0µ
oli
Iso
M]
n[
pro
10
ter
0µ
en
M]
ol
[10
0µ
M]
LP
A[
20
NE
µM
CA
]
[20
µM
Bu
]
ffe
rA
lon
e
-12
40
100
µM
]
0
40
60
µM
]
20
40
60
120
µM
]
40
Buffer Automated
Absolute Response (pm)
60
ATP Automated
Absolute Response (pm)
ATP (M anual)
60
Absolute Response (pm)
ATP (Automated)
80
80
60
hMSC (2000 c/w)
140
HUVEC 12000 c/w
80
Label-Free Response with Ion Channel Ligands
in hMSC (2000 c/w)
Label-free Ion Channel Studies In hMSC
100
hMSC 2500 c/w
80
100
100
Cell-Based Assays
Epic® technology measures
changes in light refraction
resulting from dynamic mass
redistribution (DMR) within the
cell which occurs in response to
receptor activation or
deactivation in a zone within the
cell’s monolayer. The change is
indicated by a change in
wavelength.
Label-free Receptor Panning in HUVEC Cultures
Response (pm)
100
Label-free Ion Channel Receptor Panning
µM
]
Comparison of JANUS Automated vs. Manual
Assays on hMSC (2500 c/w)
5
Label-free GPCR Receptor Panning
Label-free Receptor Panning in hMSC Cultures
JANUS Automation vs. Manual Label-free
Measurements of ATP Activation in hMSC
Response (pm)
There is growing demand for more physiologically-relevant
assay platforms, leading to increased adoption of both label-free
technologies and stem cells in research. Automated systems are
also needed to facilitate research by increasing cell-based assay
efficiency, reproducibility, and performance. To address these
needs, methods utilizing the PerkinElmer EnSpire™ Multimode
Plate Reader with Corning® Epic® label-free technology and the
JANUS® Automated Workstation have been developed to noninvasively identify and characterize multiple Ion Channel and
GPCR activation in Lonza® CloneticsTM Human Umbilical Vein
Endothelial Cells (HUVEC) and PoieticsTM human
Mesenchymal Stem Cells (hMSC). By monitoring the ligandinduced dynamic mass redistribution (DMR) in living cells with
no need for cellular or ligand modification, the EnSpire labelfree platform detects cellular responses from endogenously
expressed receptors and ion channels. This obviates the need to
engineer cells to over-express receptors of interest, thus greatly
reducing the possibility of altering cellular biology. These data
demonstrate the applicability of label-free technology for
primary and stem cell research and the utility of liquid handling
automation to ensure highly reproducible data from these
primary cell assays.
4
Automating Stem Cell Assays
Do
pa
mi
2
Abstract
Response (pm)
1
Histamine
Clobenpropit
EC50
2.377e-006
~ 0.2982
ATP
Suramin
EC50
1.158e-006
0.01510
Testing the specificity of the agonist response using antagonists in the
hMSC Purinergic and Histamine pathways.
• Suramin and Clobenpropit clearly inhibit ATP and Histamine agonist
activity, respectively.
• This inhibition confirms that Purinergic and Histamine receptor activation
can be robustly measured using the EnSpire label-free cellular assay.
The EnSpire Label-free Platform Offers:
• A versatile tool for pathway-independent,
global analysis of basic systems biological
research and physiologically-relevant drug
discovery.
• A non-invasive tool to detect GPCR and Ion
channel activation in stem and primary cell
types.
o Suggesting it is a viable platform for
studying embryonic stem cells and derived
cell types.
• In conjunction with the JANUS AWS,
significant reduced cost, assay development
time and reagents requirements for primary
and stem cell research.
• In collaboration with the high quality and fully
optimized Lonza products, a sensitive and
robust system that easily detects both
endogenously and recombinantly expressed
receptors in receptor panning experiments.
PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com