Copyright © 2020 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License jove.com June 2020 • 160 • e61455 • Page 1 of 18 A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury Hussein Atta *,1,2 , Ali Hashemi Gheinani *,1,2 , Amanda Wacker 1 , Yaser Heshmati 3,4,5 , Alex Bigger-Allen 1,6 , George Lambrinos 1,2 , Yao Gao 2,7 , Diane R. Bielenberg 2,7 , Rosalyn M. Adam 1,2 1 Department of Urology, Boston Children's Hospital 2 Department of Surgery, Harvard Medical School 3 Division of Hematology/Oncology, Harvard Medical School Boston 4 Dana-Farber Cancer Institute 5 Broad Institute 6 Biological Biomedical Sciences Program, Division of Medical Sciences, Harvard Medical School 7 Vascular Biology Program, Boston Children's Hospital * These authors contributed equally Corresponding Authors Ali Hashemi Gheinani Ali.HashemiGheinani@childrens.harvard.edu Rosalyn M. Adam Rosalyn.Adam@childrens.harvard.edu Citation Atta, H., Gheinani, A.H., Wacker, A., Heshmati, Y., Bigger- Allen, A., Lambrinos, G., Gao, Y., Bielenberg, D.R., Adam, R.M. A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury. J. Vis. Exp. (160), e61455, doi:10.3791/61455 (2020). Date Published June 17, 2020 DOI 10.3791/61455 URL jove.com/video/61455 Abstract We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and subsequent bladder wall remodeling. To facilitate assessment of the cellular composition of the bladder wall in non-injured control and spinal cord injured mice, we developed an optimized dissociation protocol that supports high cell viability and enables the detection of discrete subpopulations by flow cytometry. Spinal cord injury is created by complete transection of the thoracic spinal cord. At the time of tissue harvest, the animal is perfused with phosphate-buffered saline under deep anesthesia and bladders are harvested into Tyrode’s buffer. Tissues are minced prior to incubation in digestion buffer that has been optimized based on the collagen content of mouse bladder as determined by interrogation of publicly available gene expression databases. Following generation of a single cell suspension, material is analyzed by flow cytometry for assessment of cell viability, cell number and specific subpopulations. We demonstrate that the method yields cell populations with greater than 90% viability, and robust representation of cells of mesenchymal and epithelial origin. This method will enable accurate downstream analysis of discrete cell types in mouse bladder and potentially other organs. Introduction Perturbations of normal urinary bladder function can lead to decreased quality of life for many individuals. In order to gain a better understanding of how injury or disease derails normal bladder function, it is important to probe