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

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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.