Name: Dissociation of CNS Tissue for Isolating Cells in EAE Mice
Uploaded: 2023-10-06T13:40:00
Duration: 1 min 35 s
Description: Watch this Scientific Journal Video about Dissociation of CNS Tissue for Isolating Cells in EAE Mice at JoVE.com

dissociation of cns tissue for isolating cells in eae mice 

Aislamiento simultáneo de todos los tipos de células residentes del SNC de ratones autoinmunes

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.

Autor destacado: Estudio de las vías neuroinflamatorias mediante el aislamiento simultáneo de todos los principales tipos de células residentes en el SNC 

Aislamiento simultáneo de los principales tipos de células residentes en el sistema nervioso central de ratones adultos con encefalomielitis autoinmune

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 Dissociation of CNS Tissue for Isolating Cells in EAE Mice at JoVE.com

Dissociation of CNS Tissue for Isolating Cells in EAE Mice   

Disociación de tejido del SNC para aislar células en ratones EAE 

Para comenzar, disecciona el tejido del cerebro y la médula espinal y almacena la suspensión celular en hielo. A continuación, prepare un volumen adecuado de mezcla de enzimas-1 y 2. Transfiera 1.950 microlitros de mezcla enzimática-1 al tubo C y agregue los trozos de tejido de la suspensión celular del SNC.A continuación, añada 30 microlitros de mezcla de enzimas-2 al tubo C. Cierre bien los tubos C y fíjelos boca abajo en el manguito del disociador de celdas con calentadores. Ejecute el programa apropiado y observe durante al menos cinco minutos para asegurarse de que todos los tubos giren a la misma velocidad.Mientras tanto, coloque un colador de 70 micras en un tubo de 50 mililitros y humedezca previamente el colador con dos mililitros de DPBS. Una vez finalizado el programa, separe los tubos C del disociador y centrifugue las muestras a 300 g durante un minuto a cuatro grados centígrados. Vuelva a suspender la muestra y aplíquela al colador previamente humedecido.Agregue 10 mililitros de DPBS frío al tubo C vacío. Mezcle y agregue la suspensión en el colador correspondiente. Después de desechar los coladores, centrifugue la suspensión celular nuevamente durante 10 minutos y luego aspire cuidadosamente el sobrenadante para obtener un gránulo celular.

Research

JoVE Journal

Neuroscience

Dissociation of CNS Tissue for Isolating Cells in 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 ...

Investigación

Neurociencias

Preparation of CNS Tissue for Isolating Cells in EAE Mice 

Preparación de tejido del SNC para aislar células en ratones EAE 

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

Eliminación de restos y glóbulos rojos de la suspensión de células del SNC de ratones EAE

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

Aislamiento de tipos celulares del SNC mediante perlas magnéticas en ratones naive y EAE