1. Phenotypic Chemotaxis by the use of
flow cytometry
A Freeman1, J Hincks1, Y Whitehead1, C Murray2, G Wilkinson2, M Fagura2
Introduction
Chemotaxis assays have been traditionally set up to analyse
gross migration of cells across a membrane in response to a
chemotractant either by manual counting or by the use of a
cellular dye measuring gross cellular movement. For simple
assay systems where a recombinant cell line is being used for
basic efficacy measurement this system works well. However,
Chemotaxis assays can also be used to monitor the migration
of specific cell types from within a mixed population of cells. At
Huntingdon Life Sciences we have worked with AstraZeneca
on the development of a phenotyping Chemotaxis assay by the
use of flow cytometry to monitor the migration of the specific
cell types within human Peripheral Blood Mononuclear Cells
(PBMCs) in response to MDC. Due to the nature of this novel
assay it then allows the use of disease state PBMC samples
(or different conditioned media from separate primary assay
systems) to be used for mechanism of action (MOA) studies with
compounds, or to allow the biological investigation of disease
state samples and the cell types involved.
Described within this poster is the development of a T Cell
Chemotaxis assay utilising flow cytometry to phenotype cellular
migration in response to MDC (macrophage derived chemokine;
CCL22), an agonist of the human CCR4 chemokine receptor. In
particular the assay has been used to specifically quantify the
numbers of CD4+/CLA+ T cells migrating as well as the effect of
inhibition of that response with an AstraZeneca CCR4 inhibitor
Assay system
The HTS Transwell-96 System is composed of four
components:
● A 96 well permeable support plate - with choice of membranes
● Reservoir plate (single well feeder plate) - with removable
media stabilizer
● A 96 well receiver plate - for use with cell growth or assay
● Lid - minimizes evaporation and protects against
contamination
Method
Transwell insert
Upper compartment
Microporous membrane
Lower compartment
Human PBMCs isolated from whole blood using Histopaque
PBMCs counted and prepared ready for T Cell isolation
CD4 T Cells isolated using a Milltenyi Biotech CD4
negative selection kit
CD4 Isolated T Cells counted
175 μl Chemotractant at desired concentrations and compound
(where required) added to the bottom wells of a 96 well Transwell plate
1.25 x 105 CD4 Cells added to the filter layer of the Transwell insert
Transwell plate incubated for 2 hours at 37°C/5% CO2
The Transwell filter is discarded and the cells in the lower wells
are stained for CD3, CD4, CD8, CLA and CD195. The appropriate
compensation controls and FMO’s are also prepared
Cell samples analysed and counted using a BD FACS
Canto II flow cytometer
Figure 2a: Concentration-effect curve for the migration of
isolated CD4+ T cells in response to MDC. Data are the mean
+/- SD of 2 determinations and are representative of 4 individual
experiments.
EC50 = 1nM
Figure 2b: Concentration-effect curve for the migration of
isolated CD4+ T cells in response to MDC in the presence or
absence of the indicated concentrations of a CCR4 antagonist.
Data are the mean +/- SD of 2 determinations and representative
of 3 individual experiments.
CLA+CCR4+ chemotaxis
800 MDC only
0.1 1 10 100
600
400
200
0
-200
3uM AZ
1uM AZ
0.3uM AZ
0.1uM AZ
0.03uM AZ
[MDC] nM
CLA+CCR4+ Cells number
Discussion
The assay described within this poster has shown the
development of a novel Chemotaxis based method to monitor
the migration of specific cell populations within PBMC samples.
Specifically we have shown the migration of CCR4+ CLA+
skin homing T cells in response to the CCR4 agonist MDC
and the inhibition of that response with an AstraZeneca CCR4
antagonist. The migration of these cells in response to MDC
yielded an EC50 of 1nM, which is in accordance with the literature
for the effect of this chemokine at the CCR4 receptor.
The efficacy of the AstraZeneca compound measured was
also consistent with the potency observed in a human CCR4
receptor-binding assay (pIC50 = 8.3). Using this assay we have
demonstrated that it is possible to accurately determine the
numbers of cells migrating in response to receptor stimuli and
suggest that this may be a useful method to quantify the effect
of such ligands, and specific inhibitors and antagonists, on the
migration of cells derived from disease samples such as atopic
dermatitis.
A major benefit of using flow cytometry to monitor the specific
cell types that have migrated compared to a measure of gross
cell migration, means that the assay system can be used as
a format for investigating cellular mechanism within disease
samples.
Conclusion
This poster describes the successful development of a
Chemotaxis method at HLS for the phenotypic analysis of the
migrated cells. Using flow cytometry with the Transwell system,
has enabled the Chemotaxis assay to not only be used as a
primary efficacy assay, but also be used as an assay system to
investigate disease mechanism.
Using flow cytometry with the Transwell system also enables:
● Rare cell types to be identified
● A cell count of cells migrated
● Phenotype of cells migrated
● Use of low sample volumes
Poster 661
Results
MDC CLA+ CCR4+
150
100
50
0
0.001 0.01 0.1 1 10 100 1000
MDC[nM]
Cell Number
Figure 1a: 0.3nM MDC
CD8+
Figure 1b: 1nM MDC
CD8+
Figure 1c: 10nM MDC
CD8+
Figure 1d: Cell migration positive control
CD8+
www.huntingdon.com 1Huntingdon Life Sciences, Huntingdon, Cambridgeshire, England. 2AstraZeneca, Macclesfield, Cheshire, England.