Name: Isolation of CNS Cell Types Using Magnetic Beads in Naive and EAE Mice
Uploaded: 2023-10-06T13:40:00
Duration: 1 min 32 s
Description: Watch this Scientific Journal Video about Isolation of CNS Cell Types Using Magnetic Beads in Naive and EAE Mice at JoVE.com

isolation of cns cell types using magnetic beads in naive and eae mice

Isolere alle CNS-residente celletyper samtidig 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: Studerer nevroinflammatoriske veier gjennom samtidig isolasjon av alle viktigste CNS-residente celletyper 

Samtidig isolering av hovedcelletyper i sentralnervesystemet fra voksne autoimmune encefalopatimus

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 Isolation of CNS Cell Types Using Magnetic Beads in Naive and EAE Mice at JoVE.com

Isolation of CNS Cell Types Using Magnetic Beads in Naive and EAE Mice  

Isolering av CNS-celletyper ved bruk av magnetiske perler i naive og EAE-mus

For å begynne, del den rensede, ufortynnede musens CNS-cellesuspensjon i to fraksjoner for ytterligere isolasjoner av mikroglia og oligodendrocytter. Deretter tilsettes magnetisk merket mikroperle spesifikt for overflateantigenet for forskjellige CNS-celletyper til den respektive cellesuspensjonen. Plasser kolonnen i magnetfeltet, lik kolonnen, og legg deretter til den resulterende cellesuspensjonen i kolonnen.Magnetisk merkede celler beholdes i kolonnen, og umerkede celler vil løpe gjennom. Etter å ha fjernet kolonnen fra magnetfeltet, skyll ut magnetisk merkede celler fra kolonnen inn i et rør som den positivt valgte cellefraksjonen. Del den negative strømmen gjennom fra oligodendrocyttene i to fraksjoner for ytterligere isolasjoner av nevroner og astrocytter.Flowcytometrianalyse ved hjelp av celletypespesifikke markører kombinert med diskriminering av levende eller døde celler ga mikroglia, oligodendrocytter, astrocytter og nevroner. Fenotypisk karakterisering av de forskjellige cellepopulasjonene viste at enkeltcellesuspensjoner med ca. 90 % renhet og levedyktighet ble oppnådd for alle CNS-residente celletyper.

Research

JoVE Journal

Neuroscience

Isolation of CNS Cell Types Using Magnetic Beads in Naive and 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 ...