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Novel kidney dissociation protocol and image-based flow cytometry facilitate improved analysis of injured proximal tubules


Manolopoulou M , Matlock BK , Nlandu Khodo S , Simmons AJ , Lau KS , Phillips-Mignemi M , Ivanova A , Alford CE , Flaherty DK , Gewin LS , . American journal of physiology. Renal physiology. 2019 2 13; ().


Flow cytometry studies on injured kidney tubules are complicated by a low yield of nucleated single cells. Furthermore, cell-specific responses such as cell cycle dynamics in vivo has conventionally relied on indirect immunohistochemistry and proximal tubule markers that may be downregulated in injury. We report a new tissue dissociation protocol for kidney with an early fixation step that greatly enhances the yield of single cells. Genetic labeling of the proximal tubule with either the mT/mG “tomato” or the R26Fucci2aR (Fucci) cell cycle reporter mice allows us to follow proximal tubule-specific changes in cell cycle after renal injury. Image-based flow cytometry (FlowSight) enables gating of the cell cycle and concurrent visualization of the cells with brightfield and fluorescence. We used the Fucci mouse in conjunction with FlowSight to identify a discrete polyploid population in proximal tubules after aristolochic acid injury. The tissue dissociation protocol in conjunction with genetic labeling and image-based flow cytometry are tools that can improve our understanding of any discrete cell population following injury.