Nilesh M Agalave1, Brandon T Lane1, Prapti H Mody1, Thomas A Szabo-Pardi1, Michael D Burton2. 1. Neuroimmunology and Behavior Laboratory, School of Behavioral and Brain Sciences, Center for Advanced Pain Studies, University of Texas at Dallas, Richardson, Texas, USA. 2. Neuroimmunology and Behavior Laboratory, School of Behavioral and Brain Sciences, Center for Advanced Pain Studies, University of Texas at Dallas, Richardson, Texas, USA. Electronic address: Michael.Burton@utdallas.edu.
Abstract
BACKGROUND: Neuroimmunologists aspire to understand the interactions between neurons, microglia, and astrocytes in the CNS. To study these cells, researchers work with either immortalized cell lines or primary cells acquired from animal tissue. Primary cells reflect in vivo characteristics and functionality compared to immortalized cells; however, they are challenging to acquire and maintain. NEW METHOD: Established protocols to harvest primary glia use neonatal rodents, here we provide a method for simultaneously isolating microglia and astrocytes from brain and/or spinal cord from adult rodents. We utilized a discontinuous percoll density gradient enabling easy discrimination of these cell populations without enzymatic digestion or complex sorting techniques. RESULTS: We found cells isolated from the percoll interface between 70 %-50 % were microglia, as they express ionizing calcium-binding adaptor molecule 1 (Iba1) in immunocytochemistry and CD11bhi and CD45lo using flow cytometry. Isolated cells from the 50 %-30 % interface were astrocytes as they express glial fibrillary acidic protein (GFAP) in immunocytochemistry and Glutamate aspartate transporter (GLAST)-1 using flow cytometry. Cultured microglia and astrocytes showed a functional increase in IL-6 production after treatment of lipopolysaccharide (LPS). COMPARISON WITH EXISTING METHODS: Our method allows for rapid isolation of both microglia and astrocytes in one protocol with relatively few resources, preserves cellular phenotype, and yields high cell numbers without magnetic or antibody sorting. CONCLUSION: Here we show a novel, single protocol to isolate microglia and astrocytes from brain and spinal cord tissue, allowing for culturing and other downstream applications from the cells of animals of various ages, which will be useful for researchers investigating these two major glial cell types from the brain or spinal cord of the same rodent.
BACKGROUND: Neuroimmunologists aspire to understand the interactions between neurons, microglia, and astrocytes in the CNS. To study these cells, researchers work with either immortalized cell lines or primary cells acquired from animal tissue. Primary cells reflect in vivo characteristics and functionality compared to immortalized cells; however, they are challenging to acquire and maintain. NEW METHOD: Established protocols to harvest primary glia use neonatal rodents, here we provide a method for simultaneously isolating microglia and astrocytes from brain and/or spinal cord from adult rodents. We utilized a discontinuous percoll density gradient enabling easy discrimination of these cell populations without enzymatic digestion or complex sorting techniques. RESULTS: We found cells isolated from the percoll interface between 70 %-50 % were microglia, as they express ionizing calcium-binding adaptor molecule 1 (Iba1) in immunocytochemistry and CD11bhi and CD45lo using flow cytometry. Isolated cells from the 50 %-30 % interface were astrocytes as they express glial fibrillary acidic protein (GFAP) in immunocytochemistry and Glutamate aspartate transporter (GLAST)-1 using flow cytometry. Cultured microglia and astrocytes showed a functional increase in IL-6 production after treatment of lipopolysaccharide (LPS). COMPARISON WITH EXISTING METHODS: Our method allows for rapid isolation of both microglia and astrocytes in one protocol with relatively few resources, preserves cellular phenotype, and yields high cell numbers without magnetic or antibody sorting. CONCLUSION: Here we show a novel, single protocol to isolate microglia and astrocytes from brain and spinal cord tissue, allowing for culturing and other downstream applications from the cells of animals of various ages, which will be useful for researchers investigating these two major glial cell types from the brain or spinal cord of the same rodent.
Authors: Rosa C Paolicelli; Giulia Bolasco; Francesca Pagani; Laura Maggi; Maria Scianni; Patrizia Panzanelli; Maurizio Giustetto; Tiago Alves Ferreira; Eva Guiducci; Laura Dumas; Davide Ragozzino; Cornelius T Gross Journal: Science Date: 2011-07-21 Impact factor: 47.728
Authors: Marie Orre; Willem Kamphuis; Lana M Osborn; Jeroen Melief; Lieneke Kooijman; Inge Huitinga; Jan Klooster; Koen Bossers; Elly M Hol Journal: Neurobiol Aging Date: 2013-08-15 Impact factor: 4.673
Authors: Jason C Dugas; Wim Mandemakers; Madolyn Rogers; Adiljan Ibrahim; Richard Daneman; Ben A Barres Journal: J Neurosci Date: 2008-08-13 Impact factor: 6.167
Authors: Nilesh M Agalave; Resti Rudjito; Alex Bersellini Farinotti; Payam Emami Khoonsari; Katalin Sandor; Yuki Nomura; Thomas A Szabo-Pardi; Carlos Morado Urbina; Vinko Palada; Theodore J Price; Helena Erlandsson Harris; Michael D Burton; Kim Kultima; Camilla I Svensson Journal: Pain Date: 2021-02-01 Impact factor: 7.926