Improved flow cytometric and Western blot detection of PLXDC2 in human iPS cells via BACE-1 inhibition

PLXDC2 is a single-pass transmembrane glycoprotein expressed in multiple stem and immune-related cell types. In human induced pluripotent stem cells (hiPSCs), PLXDC2 mRNA and its corresponding protein have been confirmed by transcriptome analysis and mass spectrometry; however, surface detection by flow cytometry using the monoclonal antibody #4G3 cannot be achieved after routine TrypLE dissociation. This failure is attributable to degradation of protease-sensitive epitopes by TrypLE and membrane-associated proteases such as BACE-1 (β-site APP cleaving enzyme 1). To retain these epitopes, we evaluated a protease-free mechanical dissociation procedure in combination with BACE-1 inhibition during both cell dissociation and antibody staining. This approach enabled reproducible flow cytometric detection of PLXDC2 on 201B7 hiPSCs. Western blot analysis of membrane fractions similarly showed increased PLXDC2 signal intensity when samples were processed in the presence of a BACE-1 inhibitor. These results demonstrate a practical method for improving PLXDC2 detection in hiPSCs and provide a general strategy for analyzing cell-surface proteins that are susceptible to proteolytic cleavage.