Brain Tissue Dissociation for Cell Sorting v1 (protocols.io.nx7dfrn)

protocols.io ◽  
2018 ◽  
Author(s):  
Grant Lin ◽  
Michelle Monje
Keyword(s):  
Brain Tissue ◽  
Cell Sorting ◽  
Tissue Dissociation

scholarly journals Translational profiling of microglia reveals artifacts of cell sorting

2017 ◽  
Author(s):  
Silvia S. Kang ◽  
Kelsey E. Baker ◽  
Xuewei Wang ◽  
Jeanne-Pierre Kocher ◽  
John D. Fryer
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Cell Sorting ◽  
Local Environment ◽  
Innate Immune ◽  
Immune Challenge ◽  
Cellular Activation ◽  
Disease States ◽  
Tissue Dissociation ◽  
The Central Nervous System

AbstractMicroglia are the resident innate immune population of the central nervous system that constantly survey and influence their local environment. Transcriptomic profiling has led to significant advances in our understanding of microglia in several disease states, but tissue dissociation and purification of microglia is known to lead to cellular activation. Here we use RiboTag translational RNAseq profiling to demonstrate that commonly used cell sorting methods lead to a fundamental alteration of the microglial transcriptome, with several transcripts that can be used to mark artifacts of isolation. Microglial RiboTag RNAseq profiling after peripheral immune challenge with lipopolysaccharide demonstrates unique transcriptional targets that are not evident using cell sorting methodology. Finally, we applied our technique to reveal novel shared and distinct pathways when comparing microglial transcriptomes after peripheral challenge with bacterial or viral mimetics. This study has broad implications for approaches that examine microglial transcriptomes in normal and pathological states.SummaryKang et al. demonstrate artifactual induction of microglial transcripts associated with cell sorting. Using RiboTag translational profiling methodology, several markers of cell sorting artifact were revealed. Furthermore, RiboTag isolation unveiled changes in microglial transcriptomes following systemic inflammation that would otherwise have been masked by artifacts of cell sorting.

scholarly journals Minimizing the ex vivo confounds of cell-isolation techniques on transcriptomic profiles of purified microglia

2021 ◽  
Author(s):  
Sarah R Ocanas ◽  
Kevin D Pham ◽  
Harris E Blankenship ◽  
Adeline H Machalinski ◽  
Ana J Chucair-Elliott ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Ex Vivo ◽  
Cell Isolation ◽  
Cell Preparation ◽  
Tissue Samples ◽  
In Vivo Models ◽  
Isolation Techniques ◽  
Tissue Dissociation ◽  
Neuroscience Research

Modern molecular neuroscience studies require analysis of specific cellular populations derived from brain tissue samples to disambiguate cell-type specific events. This is particularly true in the analysis of minority glial populations in the brain, such as microglia, which may be obscured in whole tissue analyses. Microglia have central functions in development, aging, and neurodegeneration and are a current focus of neuroscience research. A long-standing concern for glial biologists using in vivo models is whether cell isolation from CNS tissue could introduce ex vivo artifacts in microglia, which respond quickly to changes in the environment. Mouse microglia were purified by magnetic-activated cell sorting (MACS), as well as cytometer- and cartridge-based fluorescence-activated cell sorting (FACS) approaches to compare and contrast performance. The Cx3cr1-NuTRAP mouse model was used here to provide an endogenous fluorescent microglial marker and a microglial-specific translatome profile as a baseline comparison lacking cell isolation artifacts. All methods performed similarly for microglial purity with main differences being in cell yield and time of isolation. Ex vivo activation signatures occurred principally during the initial tissue dissociation and cell preparation and not the microglial cell sorting. Utilizing transcriptional and translational inhibitors during the cell preparation prevented the activational phenotype. These data demonstrate that a variety of microglial isolation approaches can be used, depending on experimental needs, and that inhibitor cocktails are effective at reducing cell preparation artifacts.

An optimized method for adult zebrafish brain-tissue dissociation that allows access mitochondrial function under healthy and epileptic conditions

2021 ◽  
pp. 147498
Author(s):  
Luana Moro ◽  
Giovana Rech ◽  
Amanda Martins Linazzi ◽  
Thainá Garbino dos Santos ◽  
Diogo Lösch de Oliveira
Keyword(s):  
Brain Tissue ◽  
Mitochondrial Function ◽  
Adult Zebrafish ◽  
Zebrafish Brain ◽  
Tissue Dissociation

Magnetic cell sorting: A fast and effective method of concurrent isolation of high purity viable astrocytes and microglia from neonatal mouse brain tissue

2008 ◽  
Vol 175 (1) ◽  
pp. 108-118 ◽  
Author(s):  
Ryan Marek ◽  
Michael Caruso ◽  
Abdolmohamad Rostami ◽  
Judith. B. Grinspan ◽  
Jayasri Das Sarma
Keyword(s):  
Brain Tissue ◽  
Mouse Brain ◽  
High Purity ◽  
Cell Sorting ◽  
Neonatal Mouse ◽  
Magnetic Cell Sorting

Effects of therapeutic and toxic phenobarbital doses on brain tissue caspase 3-activity in the neonatal rat

2006 ◽  
Vol 37 (03) ◽  
Author(s):  
R Husain ◽  
HS Fink ◽  
K Lang ◽  
B Merkle ◽  
R Bauer ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Caspase 3 ◽  
Neonatal Rat
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