Abstract 6310:Ex vivophosphoflow cytometry to capture fast changes of phospho-proteins at single cell level

BACKGROUND: Protein phosphorylation is an important cellular regulatory process and a key indicator of signaling activation or deactivation. However, the fast switch-on/off of the process makes difficult the timely and accurate detection of the phosphorylation status. It is even more challenging to monitor the protein phosphorylation during cancer immune response in vivo, whereas immune responses vary among different cell types and can sometimes only be detected in certain rare populations like Treg (T regulatory) and TCM (T central memory) cells. The ability of Phosphoflow cytometry to accurately and rapidly detect many phospho-protein targets simultaneously at single cell level provides significant advantages over conventional methods. Ex vivo phosphoflow cytometry is increasingly being used as a tool for the discovery of biomarkers used in monitoring of disease and therapy progress. RESULTS: We established an ex vivo phosphoflow system to evaluate fast changes of pStat5 at single cell level in mouse models. Method validation was carried out step by step to ensure the reproducibility of this assay. The method development included optimizing gating strategy for non-phosphorylated proteins, selecting the reagents and optimizing the procedures of Lyse/Fix, perm, setting up positive controls under in vitro and ex vivo conditions, and optimizing phosphorylated protein staining and detection. Our data showed that the test articles induced differential pStat5 expression in different cell types, and in a dose-dependent manner. SUMMARY: We have established reliable ex vivo phosphoflow method to detect the fast changes of phospho-proteins at single cell level in animal models. This powerful method will enable us to examine protein post-translational modification in complicated host systems. Citation Format: Qiyao Zhang, Jingjing Wang, Yun Ge, Yajie Xiong, Beibei Qin, Qingyang Gu, Qunsheng Ji. Ex vivo phosphoflow cytometry to capture fast changes of phospho-proteins at single cell level [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6310.