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

Isolating All CNS Resident Cell Types Simultaneously from Autoimmune Mice

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.

Author Spotlight: Studying Neuroinflammatory Pathways Through Simultaneous Isolation of All Main CNS-Resident Cell Types 

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

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  

To begin, obtain a cell pellet after dissociation of CNS tissue and re-suspend it with 3, 100 microliters of DPBS in one 15-milliliter tube. Do not vortex. Add 1, 800 microliters of the debris removal solution from the adult brain dissociation kit to two pooled CNS cell suspensions.Invert the tube and mix the suspension, then gently overlay it with 4 milliliters of cold DPBS and observe a clear gradient. Centrifuge the tubes for 10 minutes at 3, 000g at 4 degrees Celsius with full acceleration and no break. After centrifugation, three phases are formed.Aspirate the two top phases completely and discard them, ensuring no myelin residues are left behind. Fill up the tube with cold DPBS up to 14 milliliters and close it. Invert the tube with force on the workbench until the cell pellet becomes detached from the bottom of the tube.Centrifuge the sample and aspirate the supernatant completely. For red blood cell removal, add 1 milliliter of the red blood cell removal solution to the cell pellet. After re-suspending the pellet, incubate the solution for 10 minutes at 4 degrees Celsius.Then add 10 milliliters of cold PB buffer, centrifuge the sample, and completely aspirate the supernatant. For cell counting, dilute the cell suspension by 50 times in PB buffer and then by 1 to 10 dilution in 0.4%Trypan Blue solution. Then determine the cell count, using an improved counting chamber.

Research

JoVE Journal

Neuroscience

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