LIVE CELL IMAGING OF CD4+ T-CELL CHEMOKINESIS OVER IMMOBILIZED CCL21

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In the mechanistic studies of PI3K regulatory subunit isoform contributions to chemokinesis, CCL21 will be immobilized on glass coverslips. WT and TdKO murine cells will be stained with CFSE or CMTMR, added to the CCL21-coated coverslips, and imaged for 30 minutes using an inverted wide-field fluorescence microscope. As a positive control for reduced motility, WT cells will be treated with pertussus toxin (inhibits class IB PI3K T-cell migration by blocking G-protein receptor signaling) for 2 hours. If a decrease in velocity in TdKO cells is observed, then this system is adequate in mimicking in vivo chemokinesis. If so, mechanistic studies studying cell polarization and PI3K lipid generation during chemokinesis will follow.