Copyright © 2021 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License jove.com January 2021 • 167 • e61599 • Page 1 of 15 Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection Gabriella Shull *,1,2 , Christiane Haffner *,3 , Wieland B. Huttner 3 , Elena Taverna 3,4 , Suhasa B. Kodandaramaiah 1,5,6 1 Department of Biomedical Engineering, University of Minnesota 2 Department of Biomedical Engineering, Duke University 3 Max Planck Institute of Molecular Cell Biology and Genetics 4 Max Planck Institute for Evolutionary Anthropology 5 Department of Mechanical Engineering, University of Minnesota 6 Graduate Program in Neuroscience, University of Minnesota * These authors contributed equally Corresponding Authors Elena Taverna elena_taverna@eva.mpg.de Suhasa B. Kodandaramaiah suhasabk@umn.edu Citation Shull, G., Haffner, C., Huttner, W.B., Taverna, E., Kodandaramaiah, S.B. Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection. J. Vis. Exp. (167), e61599, doi:10.3791/61599 (2021). Date Published January 21, 2021 DOI 10.3791/61599 URL jove.com/video/61599 Abstract A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices. Introduction This protocol describes the use of a robot to target and manipulate single cells in brain tissue slices, focusing in particular on single neural stem cells and neurons. The robot was developed to address a central question in developmental neurobiology, that is how neural stem and progenitor cells contribute to the brain morphogenesis 1,2,3,4,5 . To answer this question, one needs to label and track single neural stem cells and follow