Development of a Robust Automated 384-well
Whole Blood Flow Cytometry Assay
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Gilead Sciences, Inc.
333 Lakeside Drive
Foster City, CA 94404
Tel: (650) 522-4751
Fax: (650) 522-5899
S Wise, B Steiner, K Huynh, L Lad, N Pagratis
SLAS 2014
3rd Annual Conference & Exhibition
January 18-22, 2014
San Diego, CA
Gilead Sciences, Inc., Foster City, CA
Background
Step 2: Automation
The signaling pathways involved in basophil activation have been implicated in allergic responses, autoimmune disorders,
Manual Pipetting
(384-well format)
pharmacodynamic response to small molecule inhibitors against components of these signaling cascades. Historically,
plate formats. This is because the smaller well size (384-well format) is seen as prohibitive for effective red blood cell
lysis. Red blood cell lysis commonly done using a 10-fold excess volume of lysis buffer to whole blood to ensure complete
lysis. As basophils are a rare population, ~1% of whole blood, a minimum amount of whole blood per well is required to
yield the signal needed for a robust assay. The well size of deep well 384-well plates will not hold the volume of whole
blood needed plus the 10-fold excess of lysis buffer. While aggressive automated pipetting techniques can assist in the
red blood cell lysis, they also lead to lymphocyte destruction leaving the sample unusable. Here we present a robust and
automated 384-well whole blood basophil activation assay. The miniaturization was achieved through multiple rounds of
red blood cell lysis and washing in deep well 384-well plates using Agilent 70uL wide-bore 384-well tips. Both lymphocyte
integrity and complete red blood cell lysis are maintained as compared to the 96-well format. Inhibitors of basophil
activation perform similarly in the 384-well and 96-well formats. This miniaturization and automation allow for increased
throughput of compound testing in lead optimization efforts and reduce the FTE resources required.
Throughput
(per week)
Original
1 –1 5
compounds
96-well
Cell destruction
Cell populations
intact
Cell populations
intact
Incomplete
RBC Lysis
Maintaining lymphocyte integrity was achieved with 70 L wide bore tips (Agilent)
and by optimizing pipetting techniques.
Objective
Format
Optimized
Pipetting Methods:
Pipetting Method Development:
Step 3: Data Acquisition
Resources
Required
2 FTE
Projects
Supported
Traditional Flow Cytometer
Sheath
fluid
2
Sample
Attune (Life Technologies)
Sheath
fluid
Laser beam
New
•
1 –2 00
compounds
384-well
1 FTE
3+
Hydrodynamic Focusing
•
•
•
To increase throughput of whole blood basophil activation assay we miniaturized the existing 96-well format to a 384well format.
•
•
Methods
Basophil activation was measured in human whole blood using the Flow CAST™ kit (Buhlmann Laboratories AG,
Acoustic Focusing
•
•
•
Blue/Violet
•
•
Step 4: Quality Control
whole blood in heparin, was collected and delivered same day (AllCells, Emeryville, CA). Whole blood samples were
activated using the anti-FceRI mAb and stained with anti-CD63-FITC and anti-CCR3-PE for 20 minutes at 37°C (per well:
40µl of whole blood was mixed with 104µl of stimulation buffer, 8µl anti-FceRI mAb, 8µl Ab stain CCR3-PE/CD63-FITC).
Cells were centrifuged at 1000g for 18 minutes and 150µl/well of supernatant removed. Red blood cells were lysed
fer, incubating at room
temperature for 10 minutes, and collecting cell pellets by centrifuging for 1200 rpms for 5 minutes. Cells were washed with
Assay
Reproducibility
EC50s
384-well Vs. 96-well
surface expression on CCR3 positive cells. The percent CD63 positive cells within the gated basophil population were
determined and normalized to the DMSO (negative control) and control compound (positive control).
Step 1: Miniaturization
Add WB
Stimulate & Stain
50µl
200µl
Wash
RBC Lysis
1.5ml
96w
1 hr @ 37ºC
200µl
20 min @ 37ºC
10 min @ RT
160µl
384w
160µl
96-well
384-well
15
200
Single Point
Duplicate Points
FTE
2
1
Projects Supported
2
3+
65µl
40µl
Re-suspend
pellet in
Lysis Buffer
Throughput (# compounds/week)
Spin
Hard Spin
Effective red blood cell lysis in
the smaller well was achieved
through multiple rounds of
incubation with lysis buffer
ALPCO is the exclusive distributor for BÜHLMANN Labs in the U.S.
The FlowCAST™ kit is for Research Use Only.
Summary
2X
Remove Supernatant
Remove Supernatant
Compound plate format
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