Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry

Autor: Pedro Tranque, Felipe Rubio, Belen Calvo, Miriam Fernández
Rok vydání: 2020
Předmět:
0301 basic medicine
Glia reactivity
SSC
side-scattered light
Cell
PBS
phosphate-buffered saline
Article
lcsh:RC321-571
Flow cytometry
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
Immune system
medicine
EBSS
Earle's Balanced Salt Solution
Lymphocytes
HBSS
Hank's Balanced Salt Solution
SIP
stock solution of isotonic Percoll
lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry
Neuroinflammation
FACS
Fluorescence-activated cell sorter
CaCl2
calcium chloride
EDTA
ethylenediaminetetraacetic acid
medicine.diagnostic_test
Microglia
General Neuroscience
FSC
forward-scattered light
MgCl2
magnesium chloride
MgSO4
magnesium sulfate
ANOVA
one-way analysis of variance
Cell biology
CNS
Central Nervous System
Neuroimmunity
i.p
intraperitoneal injection
Papain
LD
lethal dose
030104 developmental biology
medicine.anatomical_structure
chemistry
Astrocytes
Collagenase
LPS
lipopolysaccharide
RT
room temperature
Percoll
030217 neurology & neurosurgery
medicine.drug
Zdroj: IBRO Reports, Vol 8, Iss, Pp 36-47 (2020)
IBRO Reports
ISSN: 2451-8301
DOI: 10.1016/j.ibror.2019.12.004
Popis: Highlights • Recovery of neural cells is higher with 30 % Percoll gradient than 30–70 %. • Papain enhances combined extraction of microglia, astrocytes and lymphocytes. • Dispase II potentiates papain action only in adult brain. • Mechanical dissociation isolates neonatal and adult astrocytes better than enzymes. • Papain + dispase II alows cell cytometry quantification of glial activation by LPS.
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.
Databáze: OpenAIRE
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