Flow Cytometry


A Step 1: Remove noise


B Step 2: Well identification


Figure 4: Automated high throughput flow cytometer. A BD Accuri C6 flow cytometer is integrated to an IntelliCyt HyperCyt sampler which in turn is integrated with a Beckman Coulter NX. Plates are positioned directly on to the HyperCyt using a plate shuttle


D Step 4: Export and analyse data


C Step 3: Identify cell populations


Figure 5: HyperCyt method for high throughput flow cytometry. The illustrated assay assesses the ability of test compounds to inhibit the upregulation of CD11b in GRO- stimulated neutrophils in whole human blood. 10ul volumes of whole blood is first treated with compound and stimulated with GRO for a further 15min before surface maker labelling, RBC cell lysis and fixation using 4% PFA. Samples are aspirated from microplate wells and delivered to a BD-Accuri C6 flow cytometer as a series of fluid volumes (~2ul each) separated by air bubbles. A four-second flush step was introduced at the end of each row of wells. The samples are detected in the flow cytometer as discrete clusters of events that appear at uniform time intervals. Each cluster represents ~2,000 cells sampled from a well. Data is then processed by IntelliCyt’s Hyperview software. A: The first step in processing the data is to remove ‘noise’ associated with bubbles. B: The second step is to associate each time separated cell cluster with its corresponding wells – well identification. In this case 96 separate peaks can be identified. The larger time interval every 12 wells corresponds to a four-second plate shake and probe flush step introduced into the sampling process. C: The third stage involves typical flow cytometry data analysis where the populations of interest are identified and appropriate gating strategy applied. D: Finally, data is exported and automatically analysed using Activity Base XE (IBDS, Guildford, UK). Graphs depict the inhibition of GRO stimulated CD11b upregulation using the CXCR2 antagonist SCH52712318


Drug Discovery World Spring 2013


47


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