Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry
The technical difficulty to isolate microglia, astrocytes and infiltrating immune cells from mouse brain is nowadays a limiting factor in the study of neuroinflammation. Brain isolation requirements are cell-type and animal-age dependent, but current brain dissociation procedures are poorly standard...
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doaj-7cb306765c89482fb798ec8a3dcc00592020-11-25T02:51:09ZengElsevierIBRO Reports2451-83012020-06-0183647Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometryBelén Calvo0Felipe Rubio1Miriam Fernández2Pedro Tranque3Corresponding author.; Neuroglia Laboratory, Research Institute for Neurological Disorders (IDINE), Medical School, University of Castilla-La Mancha (UCLM), Albacete, SpainNeuroglia Laboratory, Research Institute for Neurological Disorders (IDINE), Medical School, University of Castilla-La Mancha (UCLM), Albacete, SpainNeuroglia Laboratory, Research Institute for Neurological Disorders (IDINE), Medical School, University of Castilla-La Mancha (UCLM), Albacete, SpainNeuroglia Laboratory, Research Institute for Neurological Disorders (IDINE), Medical School, University of Castilla-La Mancha (UCLM), Albacete, SpainThe technical difficulty to isolate microglia, astrocytes and infiltrating immune cells from mouse brain is nowadays a limiting factor in the study of neuroinflammation. Brain isolation requirements are cell-type and animal-age dependent, but current brain dissociation procedures are poorly standardized. This lack of comprehensive studies hampers the selection of optimized methodologies. Thus, we present here a comparative analysis of dissociation methods and Percoll-based separation to identify the most efficient procedure for the combined isolation of healthy microglia, astrocytes and infiltrated leukocytes; distinguishing neonatal and adult mouse brain. Gentle mechanical dissociation and DNase I incubation was supplemented with papain or collagenase II. Dispase II digestion was also used alone or in combination. In addition, cell separation efficiency of 30 % and 30–70 % Percoll gradients was compared. In these experiments, cell yield and integrity of freshly dissociated cells was measured by flow cytometry. We found that papain digestion in combination with dispase II followed by 30 % Percoll separation is the most balanced method to obtain a mixture of microglia, astrocytes and infiltrated immune cells; while addition of dispase II was not an advantage for neonatal brain. These dissociation conditions allowed flow cytometry detection of a slight glial activation triggered by sublethal LPS injection. In conclusion, the enzymes and Percoll density gradients tested here affected differently resting microglia, activated microglia/macrophages, astrocytes and infiltrated lymphocytes. Also, newborn and adult brain showed contrasting reactions to digestion. Our study highlights the strength of flow cytometry for the simultaneous analysis of neuroimmune cell populations once extraction is optimized.http://www.sciencedirect.com/science/article/pii/S2451830120300017MicrogliaAstrocytesGlia reactivityLymphocytesNeuroimmunityFlow cytometry |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Belén Calvo Felipe Rubio Miriam Fernández Pedro Tranque |
spellingShingle |
Belén Calvo Felipe Rubio Miriam Fernández Pedro Tranque Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry IBRO Reports Microglia Astrocytes Glia reactivity Lymphocytes Neuroimmunity Flow cytometry |
author_facet |
Belén Calvo Felipe Rubio Miriam Fernández Pedro Tranque |
author_sort |
Belén Calvo |
title |
Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry |
title_short |
Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry |
title_full |
Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry |
title_fullStr |
Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry |
title_full_unstemmed |
Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry |
title_sort |
dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry |
publisher |
Elsevier |
series |
IBRO Reports |
issn |
2451-8301 |
publishDate |
2020-06-01 |
description |
The technical difficulty to isolate microglia, astrocytes and infiltrating immune cells from mouse brain is nowadays a limiting factor in the study of neuroinflammation. Brain isolation requirements are cell-type and animal-age dependent, but current brain dissociation procedures are poorly standardized. This lack of comprehensive studies hampers the selection of optimized methodologies. Thus, we present here a comparative analysis of dissociation methods and Percoll-based separation to identify the most efficient procedure for the combined isolation of healthy microglia, astrocytes and infiltrated leukocytes; distinguishing neonatal and adult mouse brain. Gentle mechanical dissociation and DNase I incubation was supplemented with papain or collagenase II. Dispase II digestion was also used alone or in combination. In addition, cell separation efficiency of 30 % and 30–70 % Percoll gradients was compared. In these experiments, cell yield and integrity of freshly dissociated cells was measured by flow cytometry. We found that papain digestion in combination with dispase II followed by 30 % Percoll separation is the most balanced method to obtain a mixture of microglia, astrocytes and infiltrated immune cells; while addition of dispase II was not an advantage for neonatal brain. These dissociation conditions allowed flow cytometry detection of a slight glial activation triggered by sublethal LPS injection. In conclusion, the enzymes and Percoll density gradients tested here affected differently resting microglia, activated microglia/macrophages, astrocytes and infiltrated lymphocytes. Also, newborn and adult brain showed contrasting reactions to digestion. Our study highlights the strength of flow cytometry for the simultaneous analysis of neuroimmune cell populations once extraction is optimized. |
topic |
Microglia Astrocytes Glia reactivity Lymphocytes Neuroimmunity Flow cytometry |
url |
http://www.sciencedirect.com/science/article/pii/S2451830120300017 |
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