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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在PI 3 K调节亚基同种型对化动作用的机制研究中，将CCL 21固定在盖玻片上。 WT和TdKO鼠细胞将用CFSE或CMTMR染色，加入CCL 21包被的盖玻片中，并使用倒置宽视野荧光显微镜成像30分钟。 作为运动性降低的阳性对照，WT细胞将用百日咳毒素（通过阻断G蛋白受体信号传导抑制IB类PI 3 K T细胞迁移）处理2小时。 如果观察到TdKO细胞中的速度降低，则该系统足以模拟体内化学运动。 如果是这样的话，研究细胞极化和PI 3 K脂质生成过程中的化学动力学机制的研究将遵循。

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.