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Automation and Detection of Cisbios IP-One HTRF
Assay using Laboratory Automation Workstation and
the PARADIGM Detection Platform from Beckman
Coulter
Amy Yoder, Keith Roby, James Barry, Laura Pajak,
Ph.D, Beckman Coulter, Inc. Sharon Soh, Chris
Harbert, Francois Degorce, Cisbio International
Cell Dispense Automation Protocol User Interface
Standard The user is able to customize the
number of columns, and the cell and media volume
desired (Figure 6A). This method takes
approximately 10-20 minutes to seed an entire
plate. Custom This allows users to vary the
number of cells across the plate. The user sets
up a custom worklist that will dictate specific
volumes for each well. Custom cell dispenses can
take a longer time to complete. Reservoir Volume
Calculator This step informs the user on how
much reagent should be placed in each of the
reservoirs (Figure 6B)

• Abstract
• The IP-One HTRF Assay is a homogeneous
  competitive immunoassay which can be used to
  monitor Gq-coupled GPCR activity through the
  inositol phosphate cascade. Inositol phosphate 1
  (IP1) is a downstream metabolite of IP3 which
  accumulates in cells in the presence of LiCl
  following Gq-mediated phospholipase C (PLC)
  activation.
• Beckman Coulter has worked in collaboration with
  Cisbio to adapt the IP-One HTRF assay to
  automation workstations. The assay was automated
  using the Biomek NXP Laboratory Automation
  Workstation. Detection and analysis was
  performed using the PARADIGM Detection Platform
  with the Cisbio approved HTRF detection cartridge
  developed by Beckman Coulter. The target in this
  study is the muscarinic m1 receptor,
  overexpressed in a CHO model system, using
  carbachol and acetylcholine as reference
  agonists.
• This poster describes
• The automated workstations utilized to process
  the cellular assay.
• The results obtained from the HTRF IP-One assay
  using the PARADIGM Detection Platform and
  laboratory automation.

• Dose-response Curves
• A CHO-M1 cell line overexpressing the muscarinic
  m1 receptor was used for all experiments, and was
  plated into a 384-well tissue culture plate using
  the Cell Dispense Method. Carbachol and
  acetylcholine were used as reference agonists for
  the IP-One Assay. The assay plate was set up so
  that each column is a replicate, and includes a
  serial dilution of the agonist. The first two
  rows of the plate include a negative control with
  no agonist and a control lacking the IP1-d2
  conjugate. The no agonist control is used to
  normalize reader data. Cells are not plated in
  the final two columns which are reserved for
  duplicates of the serially diluted calibrator
  control and duplicates of the IP1 internal
  control. Delta F is used for the comparison of
  data generated on different days and reflects the
  signal to background of the assay. The negative
  control is used as the internal assay control.
  The calculation uses the formula illustrated
  below.
• Carbachol Five replicates of conditions with
  half log dilutions of carbachol were run using
  adherent cells plated at 40,000 cells per well
  and incubated overnight. The starting
  concentration for the half log serial dilution
  was 1 mM. The replicates closely overlapped, and
  gave an average logEC50 of -6.71 0.05 (195 nM)
  with a coefficient of variation of 0.5 (Figure
  8A).
• Acetylcholine Five replicates of conditions of
  half log dilutions of acetylcholine were run
  using 80,000 cells per well suspended in 7 µL of
  Stimulation Buffer. The starting concentration
  for the half log serial dilution was 25 µM. The
  average EC50 was -5.98 0.04 (10 nM) with a
  coefficient of variation of 0.3 (Figure 8B).

Detection on the PARADIGM Platform HTRF Detection
Cartridge Homogenous Time Resolved Fluorescence
cartridge designed for Cisbios HTRF technology
(Figure 3). Multimode Analysis Software v3.1
provides preconfigured HTRF measurement protocols
that automatically calculate the Cisbio
ratio. Read Parameters
Data Reduction

Ratio 665 nm/616 nm x
10,000
A) B)
Figure 6 A) User Interface for Cell Dispense. B)
Reservoir Volume Calculator for Cell Dispense
Method
Figure 3 HTRF Detection Cartridge for the
PARADIGM Detection Platform
IP-One HTRF Assay1 The IP-One assay monitors the
accumulation of myo-inositol phosphate (IP1) in
cultured cells. Typically, IP1 is quickly
converted to myo-inositol, but in this assay,
this step is blocked by the addition of LiCl in
the Stimulation Buffer. IP1 accumulates in the
cell proportionally to the receptor activation
(Figure 4). Once the cells are lysed, the
accumulated cellular IP1 and the IP1-d2 conjugate
competitively bind to anti-IP1 cryptate. When
bound, Anti-IP1 Cryptate and IP1-D2 fluoresce by
Fluorescence Resonance Energy Transfer. 1For
research use only. Not for use in diagnostic
procedures.
IP-One Automated Method User Interface The
user is able to choose a dose-response
experiment or a set of custom agonist
concentrations in addition to the number of
columns to process. When running a standard
dose-response experiment, the user can define the
dilution factor that will be used for the serial
dilution of the agonist (Figure 7A). For a
single full 384-well plate, the method takes
approximately 2 hours and 30 minutes, including
incubation times and detection. Reservoir Volume
Calculator The interface indicates the
necessary reagent volumes and placement in the
reservoir and tubes for the assay (Figure 7B).
The volumes are based on the number of samples
and are designed to minimize waste.

• Introduction
• This poster describes the automation of the
  IP-One HTRF Assay Kit on the Biomek NXP
  workstation with a Span-8 pod. The process
  includes
• IP-One HTRF Assay
• Competitive immunoassay based on monitoring of
  IP1 accumulation through Gq receptor activation
  of the inositol phosphate cascade.
• Easily and accurately miniaturized and suited for
  automation.
• Automated Cell Dispensing
• Dispenses cells and media into a 384-well tissue
  culture plate based on user defined volumes.
• Can set up adherent cells for overnight growth
  prior to processing, or can set up cells
  suspended in the IP1 stimulation buffer for
  immediate processing via the IP-One Assay.
• Automated IP-One Assay
• Uses the cell plate created by the Cell Dispense
  Method.
• Able to create serial dilution of agonist for
  dose-response experiments.
• Able to run customized experiment with user
  defined agonist concentrations
• Automated processing of the calibrator samples
  and kit control.
• Able to process three full 384-well assays in a
  single day.

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Figure 8 Curves were fit with GraphPad Prism
Software and modeled to a variable slope of
log(agonist) vs. response. A) dose-response
curve of serially diluted carbachol. B)
dose-response curve of serially diluted
acetylcholine.
Figure 4 A) HTRF IP-One Assay Overview. B)
Chemistry behind the HTRF IP-One Assay.

• Automation Overview
• Cell Dispense Method Cells and media are
  dispensed into a single 384-well plate based on
  users preferences. Users can prepare a plate of
  adherent cells which requires overnight
  incubation whereas suspension cells are dispensed
  with stimulation buffer for immediate use in the
  IP-One assay (Figure 5).
• HTRF IP-One Method Agonist and controls are
  diluted and added to cells along with stimulation
  buffer in a single 384-well cell plate. A pause
  prompts the user to remove the cell plate and
  incubate the plate offline in a cell culture
  incubator. If the user wants to do multiple
  agonists or custom concentrations, they can
  define the agonist transfer step in a worklist.
  After the offline incubation step, the method
  continues with lysis and adds conjugates to the
  plate. A second incubation is completed on the
  workstation deck, followed by detection on the
  integrated PARADIGM Platform.

Standards and Controls Standards are serially
diluted with stimulation buffer during the HTRF
IP-One method on the Biomek NXP. Standards are
run with every assay, and data from several days
was compiled to compare variability between runs.
The curves were fit with a sigmoidal
dose-response model in GraphPad Prism software,
and the EC50 of each run was calculated from this
fit. The average logEC50 for all runs was -6.28
0.06 (525 nM) with a 1.3 Coefficient of
variation between runs (Figure 9). The expected
logEC50 for this assay is -6.3 (500nM). Controls
are added in duplicate to each run, and the
response from several experiments was compiled to
compare variability between runs. The
coefficient of variation between seven different
runs was 3.6 (Table 1).
Figure 7 A) User Interface for the HTRF IP-One
assay. B) Reservoir Volume Calculator
Results Cell Dispensing CHO-M1 cells were
plated into tissue culture-treated plates using
the Cell Dispense Method at a density of 40K
cells per well in 80 µL, the cells were then
incubated 22 hours and images were taken of the
adherent cells (Figure 10). Viability was
determined qualitatively using trypan blue stain
and by visual inspection, all wells appeared to
be gt 90 viable after overnight incubation. For
suspension cells, viability was tested by plating
80K cells per well, followed by staining with
trypan blue, and counting on a manual
hemocytometer. Cells were plated by hand as a
reference for cell viability and well-to-well
variability. The starting viability of the cell
culture suspension was 94.9 1.4 and viability
after the Cell Dispense Method was 94.5 2.0.
Materials and Methods Biomek NXP workstation with
integrated PARADIGM Detection Platform The assay
was automated on the Span-8 Biomek NXP
workstation with an integrated PARADIGM Detection
Platform (Figure 1). There are two methods
necessary for total automation of the IP-One
assay. The first method is Cell Dispense which
prepares a single cell plate for the IP-One
Assay. The second method automates the IP-One
assay. This method includes adding agonists and
standards (with appropriate dilutions), adding
assay reagents and reading on the integrated
PARADIGM Detection Platform with the HTRF
Detection Cartridge. The methods were developed
using a 4x3 Automated Labware Positioner (ALP),
but can be mapped onto most Biomek NXP
workstation deck configurations (Figure 2).

Figure 5 Overview of entire automation process
for the HTRF IP-One Assay. The process begins
with cell plating on the Biomek NXP workstation.
Adherent cells are incubated overnight and the
culture media removed on Day 2. Suspension cells
are immediately ready for the HTRF IP-One Assay.
Agonists and controls are diluted and added to
the cell plate, followed by an offline incubation
for cell stimulation. The next phases of the
method includes cell lysis and conjugate addition
which includes an online incubation followed by
detection on the PARADIGM platform.
Figure 10 CHO-M1 Cells plated using the Cell
Dispense method pictured after overnight
Incubation.
Figure 1 Biomek NXP workstation with integrated
Paradigm Detection Platform.
IP-One High Throughput Assay Quality
(Z-factor) The quality of the IP-One Automated
Method was tested by calculating the Z-factor
(Z). This test is used to determine the power
of a high throughput assay by testing the ability
to distinguish significant differences between
the negative controls and samples. A value
between 0.5 and 1.0 indicates an excellent assay.
The tests were run on two separate days using
80K cells per well. The agonist used for this
experiment was carbachol and the concentrations
were approximately at the EC25 (636 nm) and EC70
(4 µM). Using the custom setting within the
IP-One Method, the sample size was 48 for each
concentration. Z for samples at EC25 and EC70
were 0.83 and 0.77 (Table 2).
Table 1 IP1 Controls comparisons from seven
assays.
Figure 9 IP1 Standard curve and EC50 comparisons
from seven assays.
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• Conclusion
• The HTRF IP-One Assay was successfully automated
  on the Biomek NXP workstation and provides low
  CVs, good reproducibility from assay to assay,
  and Z greater than 0.5
• The Cell Dispense Method provides a fast and
  efficient process for plating cells in a 384-well
  plate while maintaining cell viability.
• The combination of the two methods provides a
  flexible, automated solution for the IP-One
  Assay.

Figure 2 Deck Configuration of the Biomek NXP
workstation. A) The Cell Dispense Instrument
Setup includes P200 Barrier Tips, a lidded
Greiner 384-Well CellStar Plate, and a 12-column
reservoir for cells and media. B) The IP-One
Instrument Setup includes 1 box each of P50 Tips
and P200 Tips, a Quarter Modular Reservoir for
reagents, and the Cell Plate from the Cell
Dispense Method. The agonist, standards, and
kit control are placed in a microfuge tube rack,
and a Costar 96-Well V-Bottom plate is used for
serial dilutions. When using custom agonist
concentrations, 4 additional boxes of P50 tips
are required.
Table 2 Z factor scores for IP-One automated
method using two concentrations of carbachol. n
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