Flow Cytometry


tubes slightly improves the throughput of sample tubes. But the arrival of microwell plate-based sampling flow cytometers are key to usage in more routine applications of drug discovery. This also allows the use of further integrated robotics, peripheral automation and liquid handling automation associated with a typical pharma drug screening laboratory.


Figure 1


Beckman Coulter MPL flow cytometer integrated with a Robotic Plate handler (Beckman Coulter) and a


refrigerated stacking incubator (Thermo)


off-target effects at the single cell level5. Flow cytometry is used during clinical testing in order to assess both safety (eg anti-drug antibody testing) and pharmacokinetic assessments to monitor the plasma levels of protein and peptide based thera- peutics6.


While flow cytometry has found many applica- tions in several stages of drug discovery, its routine widespread use for high throughput drug screening has been somewhat limited. This is unlike the anal- ogous technology – automated high content imag- ing (HCI), which in terms of drug screening is an established technology common in screening labo- ratories7. The failure of flow cytometry to keep pace with HCI is largely down to low individual sample throughput on commercially available instruments. Most flow cytometers use a single tube sampling mechanism and are largely used for immune phenotyping of single (patient) clinical samples8. Automated (bar-coded) tube sampling using carousels/racks, with capacities of up to 40


ASSAY pAKT pERK


pSTAT6 CD69


TISSUE STIMULANT CELL TYPE/SPECIES Expanded T cell aCD3/aCD28 Expanded T cell aCD3/aCD28 PBMCs


IL4 Blood/PBMCs


polymerisation granulocytes CD11b


aIgM


Actin Blood/PBMCS/ IL8/GRO PBMCs


T cells/human T cells/human T cells/human


B cells/human/mouse Neutrophils/human


IL8, GRO, MCP1 Neutrophils, monocytes


Table 1: Examples of some flow cytometry assays used for target-based drug discovery at GSK 44


The increasing use of phenotypic cell-based assays in drug discovery9 has led to a drive to find ways of increasing flow cytometry throughput. At GSK we worked with Beckman Coulter to auto- mate an FC500 MPL flow cytometer (Figure 1). The flow cytometer was integrated with an auto- mated refrigerated plate stacker and automatic transfer system. This configuration has been suc- cessfully utilised for target-based drug discovery where several flow cytometry-based mechanistic and functional assays were used to profile hun- dreds of compounds in concentration-response profiling. Table 1 lists some flow cytometry assays that we used for drug screening using an automat- ed FC500. These assays were routine and run weekly over long periods of time during lead opti- misation campaigns. Such assays would be impos- sible to prosecute without appropriate facilities, peripheral automation and liquid handling. This included an on-site blood donation unit, allowing access and delivery of fresh human blood and the ability to handle it effectively and safely. One assay run routinely was used to identify antagonists of actin polymerisation in granulocytes (Figure 2). Inhibition of cell migration is the ration- ale behind a number of anti-inflammatory drug dis- covery programmes, with neutrophil chemotaxis assays often used as an assay to monitor cellular drug activity. Actin polymerisation can be used as a surrogate measure of cell chemotaxis10, and moni- toring the inhibition of granulocyte actin polymeri- sation in human whole blood offers considerable time and cost savings over conventional cell migra- tion assays. Additionally, automation is a key factor in the measurement of actin polymerisation. This is because actin polymerisation occurs within seconds following stimulation10 and in this assay was opti- mal after 45 seconds of stimulation with IL-8. Scheduling software on a Biomek FX automated liquid handler was crucial to ensure reproducibility and concomitant increased throughput.


High Throughput Flow Cytometry (HTFC)


While the use of automated plate-based flow cytometry has been successfully applied to smaller target-based assays, it has not until recently


Drug Discovery World Spring 2013


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