Development of a Robust Automated 384-well Whole Blood ...
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Development of a Robust Automated 384-well Whole Blood Flow Cytometry Assay S Wise, B Steiner, K Huynh, L Lad, N Pagratis Gilead Sciences, Inc., Foster City, CA Summary Background Step 4: Quality Control Step 1: Miniaturization Step 2: Automation Step 3: Data Acquisition Objective Methods Gilead Sciences, Inc. 333 Lakeside Drive Foster City, CA 94404 Tel: (650) 522-4751 Fax: (650) 522-5899 The signaling pathways involved in basophil activation have been implicated in allergic responses, autoimmune disorders, 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. Basophil activation was measured in human whole blood using the Flow CAST™ kit (Buhlmann Laboratories AG, 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 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). © 2014 Gilead Sciences, Inc. All rights reserved. SLAS 2014 3 rd Annual Conference & Exhibition January 18-22, 2014 San Diego, CA Scan this QR code to link to this poster and to download a PDF copy. You will be prompted to enter the following passcode: Wise Original New 96-well 384-well Format Throughput (per week) Resources Required Projects Supported 1 –1 5 compounds 1 –2 00 compounds 2 FTE 1 FTE 2 3+ • To increase throughput of whole blood basophil activation assay we miniaturized the existing 96-well format to a 384- well format. Traditional Flow Cytometer Laser beam Sheath fluid Sheath fluid Sample Attune (Life Technologies) Acoustic Focusing • • • Blue/Violet • • Hydrodynamic Focusing • • • • • Assay Reproducibility EC50s 384-well Vs. 96-well 96-well 384-well Throughput (# compounds/week) 15 200 Compound plate format Single Point Duplicate Points FTE 2 1 Projects Supported 2 3+ RBC Lysis 96w 384w Add WB Wash 20 min @ 37ºC 10 min @ RT Hard Spin Remove Supernatant Re-suspend pellet in Lysis Buffer 1 hr @ 37ºC Spin 50μl 200μl 1.5ml 200μl 40μl 160μl 160μl 65μl Stimulate & Stain 2X Remove Supernatant Effective red blood cell lysis in the smaller well was achieved through multiple rounds of incubation with lysis buffer Maintaining lymphocyte integrity was achieved with 70 L wide bore tips (Agilent) and by optimizing pipetting techniques. Manual Pipetting (384-well format) Cell destruction Incomplete RBC Lysis Optimized Pipetting Methods: Cell populations intact Pipetting Method Development: Cell populations intact ALPCO is the exclusive distributor for BÜHLMANN Labs in the U.S. The FlowCAST™ kit is for Research Use Only.
Transcript of Development of a Robust Automated 384-well Whole Blood ...
Development of a Robust Automated 384-well Whole Blood Flow Cytometry Assay
S Wise, B Steiner, K Huynh, L Lad, N Pagratis
Gilead Sciences, Inc., Foster City, CA
Summary
Background
Step 4: Quality Control
Step 1: Miniaturization
Step 2: Automation
Step 3: Data AcquisitionObjective
Methods
Gilead Sciences, Inc.333 Lakeside Drive
Foster City, CA 94404Tel: (650) 522-4751
Fax: (650) 522-5899
The signaling pathways involved in basophil activation have been implicated in allergic responses, autoimmune disorders,
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.
Basophil activation was measured in human whole blood using the Flow CAST™ kit (Buhlmann Laboratories AG,
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
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).
© 2014 Gilead Sciences, Inc. All rights reserved.
SLAS 20143rd Annual Conference & ExhibitionJanuary 18-22, 2014San Diego, CA
Scan this QR code to link to this poster and to download a PDF copy. You will be prompted to enter the following passcode: Wise
Original
New
96-well
384-well
Format Throughput(per week)
ResourcesRequired
ProjectsSupported
1 –1 5 compounds
1 –2 00compounds
2 FTE
1 FTE
2
3+
• To increase throughput of whole blood basophil activation assay we miniaturized the existing 96-well format to a 384-well format.
Traditional Flow Cytometer
Laser beam
Sheathfluid
Sheathfluid
Sample
Attune (Life Technologies)
Acoustic Focusing•••
Blue/Violet••
Hydrodynamic Focusing•••
••
Assay Reproducibility
EC50s 384-well Vs. 96-well
96-well 384-well
Throughput (# compounds/week) 15 200
Compound plate format Single Point Duplicate Points
FTE 2 1
Projects Supported 2 3+
RBC Lysis
96w
384w
Add WB Wash
20 min @ 37ºC 10 min @ RT
Hard Spin
Remove Supernatant
Re-suspend pellet in
Lysis Buffer
1 hr @ 37ºC
Spin
50µl 200µl
1.5ml
200µl
40µl
160µl 160µl65µl
Stimulate & Stain
2X
Remove Supernatant
Effective red blood cell lysis in the smaller well was achieved
through multiple rounds of incubation with lysis buffer
Maintaining lymphocyte integrity was achieved with 70 L wide bore tips (Agilent) and by optimizing pipetting techniques.
Manual Pipetting(384-well format)
Cell destruction Incomplete RBC Lysis
Optimized Pipetting Methods:
Cell populationsintact
Pipetting Method Development:
Cell populationsintact
ALPCO is the exclusive distributor for BÜHLMANN Labs in the U.S. The FlowCAST™ kit is for Research Use Only.
