Flow Cytometry


A


B


C


Figure 2: Neutrophil actin polymerisation monitored using flow cytometry. Cells were incubated with compound for 30 minutes at 37˚C and stimulated with IL-8 for 45 seconds at room temperature. Cells are fixed, permeabilised and stained using L-alpha-lysophosphoatidyl-choline (Sigma), 4% PFA and Alexa 488 phalloidin (invitrogen) for 30min at room temperature in the dark, before analysis using flow cytometry. A: Flow cytometry light scattering plots and fluorescence histogram overlays plots depicting an increase in fluorescence intensity following actin polymerisation. B: Plots showing the effects of IL8 on both neutrophil chemotaxis and actin polymerisation. Similar EC50s of 9.6 and 9.4 in both chemotaxis and actin polymerisation were obtained. C: Various inhibitor effects on both IL8 stimulated chemotaxis and actin polymerisation, showing a close correlation between the two assays


approach of integrating the system with a Beckman Coulter Biomek NX. We found that this was more suitable than a conventional robotic arm as it allowed us to perform just-in-time additions to plates as well as providing extra lab liquid han- dling capacity (Figure 4).


The HyperCyt can sample both 96 and 384 well A B


plates in less than four and 12 minutes respective- ly. The plate sampling time is highly dependent on the cell concentration. Plate read times as low as four minutes can be obtained when the cell number is not limiting, for example, when using immor- talised cells. However, sampling times are higher when analysing rarer sub populations of cells from


Figure 3: IntelliCyt HyperCyt sampler for flow cytometers (a) The HyperCyt principle (b) Figure from IntelliCyt 46 Drug Discovery World Spring 2013


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