Name: Removal of Debris and Red Blood Cells from CNS Cell Suspension of EAE Mice
Uploaded: 2023-10-06T13:40:00
Duration: 1 min 58 s
Description: Watch this Scientific Journal Video about Removal of Debris and Red Blood Cells from CNS Cell Suspension of EAE Mice at JoVE.com

removal of debris and red blood cells from cns cell suspension of eae mice

Isolering af alle CNS-residente celletyper samtidigt fra autoimmune mus

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) characterized by demyelination, axonal damage, gliosis, and neurodegeneration. Despite numerous research approaches in this field, the pathophysiology of MS is still not fully understood1,2,3,4. The most common animal model for investigating MS is the myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55)-induced experimental autoimmune encephalomyelitis (EAE) which shares many of its clinical and pathophysiological features5,6,7,8,9. It is based on the response of the immune system against CNS-specific antigens leading to inflammation, demyelination, and neuroaxonal degeneration. Experimental autoimmune encephalomyelitis (EAE) is a suitable model for the investigation of neuroinflammatory pathways and signaling cascades found in MS.
Current therapy options for MS are only partially effective and focus primarily on the initial inflammatory phase of the disease. However, the neurodegenerative component of MS seems to be the major challenge for long-term therapeutic approaches. Therefore, reproducible, and precise cell isolation protocols are required to investigate molecular and cellular mechanisms in autoimmune diseases in a comprehensive way. Even if some protocols for the isolation of one single cell type exist10,11,12,13,14,15, there is an unmet need for the simultaneous isolation of several CNS-resident cell populations at once. Previous protocols for the isolation of CNS-resident cells lack in preserving cellular functionality and purity, resulting in co-cultivation with neighboring cells16,17,18 or the unsuitability for complex analyses of intracellular networks ex vivo19,20,21,22.
The aim of this protocol was to establish a reproducible and comprehensive method for the simultaneous isolation of pure viable single-cell suspensions of all principal CNS-resident cell types applicable in adult healthy and EAE mice. The different cell types were isolated using magnetic-activated cell sorting (MACS)23. The cell separation can be accomplished either by positive selection, i.e., magnetic labeling of cell-type-specific surface markers, or by negative selection via biotinylation and depletion of all undesired cells. Flow cytometry was applied to ensure a purity of above 90% and a viability of at least 80% of the isolated single-cell suspensions.
In conclusion, the main goal was to establish a protocol for the simultaneous isolation of all main CNS-resident cell types as a versatile tool for the investigation of neuroinflammatory pathways offering a comprehensive and precise analysis of complex cellular networks and biochemical signaling cascades in healthy and EAE mice.

Forfatter Spotlight: Undersøgelse af neuroinflammatoriske veje gennem samtidig isolering af alle vigtigste CNS-residente celletyper 

Samtidig isolering af primære celletyper hjemmehørende i centralnervesystemet fra voksne autoimmune encefalomyelitismus

To investigate molecular and cellular mechanisms in autoimmune diseases such as multiple sclerosis, the generation of reproducible and sophisticated cell isolation protocols is an unmet need. Most of the current technologies to isolate and analyze CNS-resident cells show serious shortcomings, such as focusing on only one cell type or only postnatal mice. The current protocol for the simultaneous isolation of all main CNS-resident cell types from one CNS replicate offers the possibility to analyze complex neuronal networks and new inflammatory pathways, ex vivo from one single cell suspension, applicable to healthy mice and those with experimental autoimmune encephalomyelitis.Additionally, mice numbers are decreased Our protocol for the simultaneous isolation of all four major CNS-resident cell types in healthy and EAE mice could be used as a helpful tool for research groups studying new inflammatory pathways, allowing a much more accurate analysis of complex biomolecular mechanisms and cellular networks ex vivo. New scientific questions that could be answered with the help of our protocol are dynamic investigations in EAE during different stages of disease course, like neuroinflammation, neurodegeneration, and remission. Also, cell-cell interactions and biochemical pathways could be studied on an individual level.Likewise, functional assays could be performed with cultivated cells. We intend to focus on dynamic studies of the EAE course for multi-omic analysis from one individual CNS homogenate per EAE time point.

Watch this Scientific Journal Video about Removal of Debris and Red Blood Cells from CNS Cell Suspension of EAE Mice at JoVE.com

Removal of Debris and Red Blood Cells from CNS Cell Suspension of EAE Mice  

Fjernelse af snavs og røde blodlegemer fra CNS-cellesuspension af EAE-mus

Til at begynde med, få en cellepellet efter dissociation af CNS-væv og re-suspendere det med 3.100 mikroliter DPBS i et 15 ml rør. Må ikke hvirvel. Tilsæt 1.800 mikroliter af affaldsfjernelsesopløsningen fra det voksne hjernedissociationssæt til to poolede CNS-cellesuspensioner.Vend røret og bland suspensionen, overlejr det forsigtigt med 4 ml kold DPBS og observer en klar gradient. Centrifuger rørene i 10 minutter ved 3.000 g ved 4 grader Celsius med fuld acceleration og ingen pause. Efter centrifugering dannes tre faser.Opsug de to øverste faser helt, og kassér dem, og sørg for, at der ikke efterlades myelinrester. Fyld røret op med koldt DPBS op til 14 ml og luk det. Vend røret med kraft på arbejdsbordet, indtil cellepillen løsner sig fra bunden af røret.Prøven centrifugeres og supernatanten suges fuldstændigt. Til fjernelse af røde blodlegemer tilsættes 1 ml af opløsningen til fjernelse af røde blodlegemer til cellepelleten. Efter resuspendering af pelleten inkuberes opløsningen i 10 minutter ved 4 grader Celsius.Derefter tilsættes 10 ml kold PB-buffer, prøven centrifugeres, og supernatanten suges fuldstændigt. Til celletælling fortyndes cellesuspensionen med 50 gange i PB-buffer og derefter med 1 til 10 fortynding i 0,4 % trypanblå opløsning. Bestem derefter celletællingen ved hjælp af et forbedret tællekammer.

Research

JoVE Journal

Neuroscience

Removal of Debris and Red Blood Cells from CNS Cell Suspension of EAE Mice

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate ...