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

Isolamento simultaneo di tutti i tipi di cellule residenti nel SNC da topi autoimmuni

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.

Autore in primo piano: Studio delle vie neuroinfiammatorie attraverso l'isolamento simultaneo di tutti i principali tipi di cellule residenti nel SNC 

Isolamento simultaneo dei principali tipi di cellule residenti nel sistema nervoso centrale da topi adulti con encefalomielite autoimmune

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  

Rimozione di detriti e globuli rossi dalla sospensione delle cellule del sistema nervoso centrale dei topi EAE

Per iniziare, ottenere un pellet cellulare dopo la dissociazione del tessuto del SNC e risospenderlo con 3.100 microlitri di DPBS in una provetta da 15 millilitri. Non vorticare. Aggiungere 1.800 microlitri della soluzione per la rimozione dei detriti dal kit di dissociazione cerebrale per adulti a due sospensioni di cellule del SNC raggruppate.Capovolgere il tubo e mescolare la sospensione, quindi sovrapporla delicatamente con 4 millilitri di DPBS freddo e osservare un chiaro gradiente. Centrifugare le provette per 10 minuti a 3.000 g a 4 gradi Celsius con la massima accelerazione e senza interruzioni. Dopo la centrifugazione, si formano tre fasi.Aspirare completamente le due fasi superiori e scartarle, assicurandosi che non rimangano residui di mielina. Riempire il tubo con DPBS freddo fino a 14 millilitri e chiuderlo. Capovolgere il tubo con forza sul banco di lavoro fino a quando il pellet cellulare non si stacca dal fondo del tubo.Centrifugare il campione e aspirare completamente il surnatante. Per la rimozione dei globuli rossi, aggiungere 1 millilitro della soluzione di rimozione dei globuli rossi al pellet cellulare. Dopo aver risospeso il pellet, incubare la soluzione per 10 minuti a 4 gradi Celsius.Quindi aggiungere 10 millilitri di tampone PB freddo, centrifugare il campione e aspirare completamente il surnatante. Per la conta cellulare, diluire la sospensione cellulare di 50 volte in tampone PB e poi di 1-10 diluizioni in soluzione di blu di tripano allo 0,4%. Quindi determinare il conteggio delle cellule, utilizzando una camera di conteggio migliorata.

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Research

JoVE Journal

Neuroscience

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

94 Views.  Heinrich Heine University Duesseldorf. Per iniziare, ottenere un pellet cellulare dopo la dissociazione del tessuto del SNC e risospenderlo con 3.100 microlitri di DPBS in una provetta da 15 millilitri. Non vorticare. Aggiungere 1.800 microlitri della soluzione per la rimozione dei detriti dal kit di dissociazione cerebrale per adulti a due sospensioni di cellule del SNC raggruppate.Capovolgere il tubo e mescolare la sospensione, quindi sovrapporla delicatamente con 4 millilitri di DPBS freddo e osservare un chiaro gradient...

Video: 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 the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

To date, protocols for the simultaneous isolation of all principal central nervous system-resident cell types from the same mouse are an unmet demand. The protocol shows a procedure applicable in na&#239;ve and experimental autoimmune encephalomyelitis mice to investigate complex cellular networks during neuroinflammation and simultaneously reduce the required mice numbers.

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

Ricerca

Neuroscienze

Preparation of CNS Tissue for Isolating Cells in EAE Mice 

Begin by placing the mouse in supine position and fixing the limbs with needles. Apply 75%ethanol on the front body of the animal. Make a longitudinal section through the skin and fascia on the abdomen and thorax.Cut the ribs parasternal and fold up the thorax to gain free access to the heart. Fix the thorax folded upwards with needles. Open the right atrium using scissors.Using a cannula, apply 20 milliliters of one-time PBS into the left ventricle to flush out the blood through the incised right atrium. Make a longitudinal section by cutting the skin on top of the head of the mouse to expose the skull and shift the skin around the head using forceps. Incise the skull with the help of a scissor along the sagittal suture.Insert the tip of the scissors along the incision line to crack open the calotte. Remove the remaining parts of the calotte with forceps so that the brain is fully exposed. Remove the brain carefully and place it into a murine brain matrix.Cut the brain into one millimeter thick sagittal slices by using a razor blade. With the help of scissors, cut the vertebral column just above the iliac crest. Insert a 20 gauge needle attached to a 20 milliliter syringe containing one-time PBS into the spinal canal.Flush the spinal cord out of the spinal canal from caudal to cranial. Cut the spinal cord into 0.5 centimeter long segments using a scalpel. Store the CNS cell suspension consisting of the brain and corresponding spinal cord in a Petri dish containing three milliliters of cold DPBS.

Preparazione del tessuto del SNC per l'isolamento delle cellule nei topi EAE 

Dissociation of CNS Tissue for Isolating Cells in EAE Mice 

To begin, dissect the brain and spinal cord tissue and store the cell suspension on ice. Then prepare an appropriate volume of enzyme mix-1 and 2. Transfer 1, 950 microliters of enzyme mix-1 into the C tube, and add the tissue pieces of the CNS cell suspension.Then add 30 microliters of enzyme mix-2 to the C-tube. Close the C-tubes tightly and attach them upside down onto the sleeve of the cell dissociator with heaters. Run the appropriate program and observe for at least five minutes to ensure all tubes turn at the same velocity.In the meantime, place a 70-micron strainer on a 50-milliliter tube and pre-moisten the strainer with two milliliters of DPBS. After termination of the program, detach the C tubes from the dissociator and centrifuge the samples at 300 g for one minute at four degrees Celsius. Resuspend the sample and apply it to the pre-moistened strainer.Add 10 milliliters of cold DPBS to the empty C-tube. Mix and add the suspension onto the corresponding strainer. After discarding the strainers, centrifuge the cell suspension again for 10 minutes, and then carefully aspirate the supernatant to obtain a cell pellet.

Dissociazione del tessuto del SNC per l'isolamento delle cellule nei topi EAE 

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.

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

To begin, divide the purified, undiluted mouse CNS cell suspension into two fractions for further isolations of microglia and oligodendrocytes. Then add magnetically labeled microbead specific for the surface antigen for different CNS cell types to the respective cell suspension. Place the column in the magnetic field, equate the column, and then add the resulting cell suspension onto the column.Magnetically labeled cells will be retained within the column, and unlabeled cells will run through. After removing the column from the magnetic field, flush out magnetically labeled cells from the column into a tube as the positively selected cell fraction. Divide the negative flow through from the oligodendrocytes into two fractions for the further isolations of neurons and astrocytes.Flow cytometry analysis using cell type specific markers combined with live or dead cell discrimination yielded microglia, oligodendrocytes, astrocytes, and neurons. Phenotypic characterization of the different cell populations proved that single cell suspensions with approximately 90%purity and viability were obtained for all main CNS resident cell types.