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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在PI3K调节亚基异构体参与化学运动的机制研究中，CCL21将被固定在玻片上。WT和TdKO小鼠细胞将被CFSE或CMTMR染色，加入CCL21涂层的盖片中，并使用倒置广场荧光显微镜成像30分钟。作为运动性降低的阳性对照，WT细胞将被百日咳毒素(通过阻断G蛋白受体信号来抑制IB类PI3K T细胞迁移)处理2小时。如果观察到TdKO细胞的速度减慢，那么这个系统就足以模拟体内的化学运动。如果是这样的话，接下来将进行关于化学运动过程中细胞极化和PI3K类脂生成的机械性研究。

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.