Methods in Molecular Biology DOI 10.1007/7651_2014_114 © Springer Science+Business Media New York 2014
Isolation of Central Nervous System (CNS) Infiltrating Cells Ilgiz A. Mufazalov and Ari Waisman Abstract Leukocyte infiltration of the central nervous system (CNS) occurs under certain pathogenic conditions and most often results in severe disorders. Therefore, the isolation and analysis of such infiltrating cell populations is necessary for elucidating the underlying pathogenic mechanisms. Here we describe a simple and straightforward protocol for cell isolation from the inflamed CNS, which combines mechanical dissociation and enzymatic degradation of the tissue. Additionally, purification by Percoll gradient centrifugation provides a great yield of the infiltrating material. The isolated cells can be further used for downstream applications such as cell sorting, cellular or molecular analysis. Keywords: CNS, EAE, Multiple sclerosis, Percoll, T cells, Blood–brain barrier, Protocol
1
Introduction The integrity of the blood–brain barrier (BBB) determines proper trafficking of nutrients and (cellular) mediators into and from the CNS parenchyma, and therefore represents a key feature of the healthy individual. However, under certain circumstances disruption of the BBB leads to severe dysfunctions. Thus, BBB breakdown has been associated, among others, with Alzheimer’s disease, Parkinson’s disease, brain infections, stroke, epilepsy, and multiple sclerosis (MS) (1). MS is a common neurodegenerative disorder that affects mainly Caucasians in high-income countries and the number of people suffering from MS has increased in the last years and reached about 2.5 millions all over the world (2). The disease progression is characterized by the presence of multiple inflammatory and demyelinating lesions within the brain and spinal cord, which result in axonal damage and consequently in clinical symptoms such as sensory and motor impairment, ataxia, spasticity, fatigue, and cognitive impairment (3). MS is believed to be of autoimmune nature and has a clear inflammatory component. Despite intensive investigations during past decades the exact mechanisms of disease onset and progression still remain unknown. To study MS in more detail an animal model termed experimental autoimmune encephalomyelitis (EAE) has been established. EAE mimics many of the MS features in rodents and, despite all its
Ilgiz A. Mufazalov and Ari Waisman
limitations, remains the best tool to study the disease in vivo. Like in MS, EAE progression is also characterized by inflammation and accumulation of infiltrating peripheral cells within the CNS. Interestingly, EAE clinical symptoms can be different according to the main site of inflammation being the brain or spinal cord (4). Therefore, CNS infiltrating cell isolation is a crucial and indispensible step for understanding the mechanisms of MS/EAE pathogenesis. Here we describe a detailed protocol for the isolation of CNS infiltrating cells from animals suffering from mouse models of the aforementioned disorders with CNS associated immune responses, such as EAE. An essential basis for the protocol is the cardiac perfusion of the animals with normal saline solution to avoid possible peripheral blood cell contamination of the CNS samples. In the second step a combination of mechanical dissociation and enzymatic degradation of the extracellular matrix proteins is implemented to achieve a single cell suspension. Further purification with the colloid Percoll is necessary to enrich for CNS infiltrating cells. Pecoll is not toxic to the cells under these conditions and does not interfere with many laboratory applications, such as electronic counting instruments or fluorescent activated cell sorting (FACS). Therefore, isolated CNS infiltrating cells may be used for further analysis and characterization. Importantly, the protocol is not designed to isolate only mononuclear CNS infiltrating cells but rather attempts to purify these cells from debris, extracellular components, and most importantly the myelin. The critical step of a discontinuous three-layer Percoll gradient leads to enrichment of leukocytes as well as CNS resident cell types (such as microglia (5)) in the interphases. Beneficially, these CNS resident cell types may be used as internal control for infiltrating cell quantification. Therefore, additional purification procedures are required to isolate specific CNS infiltrating cell subsets. Total numbers of CNS infiltrating cells are strongly correlated with EAE severity and the stage of disease progression. For example, to yield the highest amounts of T cells from the diseased CNS the isolation should be performed at the peak of disease with pooled brain and spinal cord material. Using the here described method, isolation of CNS (infiltrating) cells from mice with fully paralyzed hind limbs (score 3.5), partially (score 4) or fully (score 4.5) paralyzed front limbs routinely yields 1.5–3.5 million cells, including about 0.5 million CD90 positive T cells (Fig. 1a). Importantly, the isolated cells may be further used for downstream application, e.g. the flow cytometric analysis to distinguish infiltrated lymphocytes (CD45+CD11b), macrophages (CD45highCD11b+) and microglia (CD45lowCD11b+) (Fig. 1b). Moreover, different effector T cell subsets maybe be identified and characterized based on their characteristic cytokine expression (Fig. 1c).
Cell Isolation from CNS
Fig. 1 Isolation of CNS infiltrating cells from C57Bl/6 mice with EAE. (a) Absolute numbers of live and CD90 positive cells. Data represents CNS isolated material from 19 EAE diseased mice, with clinical scores ranging from 3.5 to 4.5, from 7 independent experiments. (b) Surface markers expression profile of CNS infiltrating lymphocytes (CD45+CD11b), macrophages and microglia represents greater amounts of inflammatory cells in EAE diseased mouse (score 4) compare to nondiseased mouse (score 0). (c) Cytokine expression profile of CNS infiltrated CD4+CD40L+ T cells isolated from a mouse with clinical score 4. Cells were stimulated ex vivo in the presence of MOG peptide and Brefeldin A for 6 h
2
Materials Prepare all equipment and materials in advance, as the time is critical for cell survival during the isolation procedure. Only digestion solution and Percoll fractions should be prepared shortly before usage.
2.1
Equipment
1. Surgical instruments: scissors, forceps, i.v. perfusion system, razor blades. 2. Suppliers: 100 mm petri dishes, 15 ml Falcon tubes, 5 ml and 10 ml medical syringes, hypodermic needles (BD): 0.9 40 mm 20 G “yellow,” 1.2 40 mm 18 G “red,” 0.9 70 mm 20 G “long yellow” (see Note 1). 3. Equipment: vortexer, centrifuge with swing-out bucket rotor, water bath.
2.2
Reagents
1. Mouse anesthesia: Isofluran (see Note 2). 2. 70 % Ethanol.
Ilgiz A. Mufazalov and Ari Waisman
3. Perfusion solution: Normal saline (0.9 % w/v NaCl) (see Note 3). 4. Phosphate buffered saline (PBS), without Calcium and Magnesium. 5. PBS, with Calcium and Magnesium. 6. 10PBS, without Calcium and Magnesium. 7. Hanks’ Balanced Salt Solution (HBSS), without Calcium and Magnesium (see Note 4). 8. Fetal calf serum (FCS). 9. Cell isolation/storage buffer (PBS/FCS): PBS supplemented with 2 % FCS. 10. Digestion solution: PBS with Calcium and Magnesium, 1.5 mg/ml Collagenase II, 50 μg/ml DNase I (see Note 5). 11. Stock Isotonic Percoll (SIP) solution: 90 % Percoll plus 10 % 10PBS, without Calcium and Magnesium (see Note 6). 12. 30 %—Percoll fraction: 30 % SIP plus 70 % PBS, without Calcium and Magnesium. 13. 37 %—Percoll fraction: 37 % SIP plus 63 % HBSS, without Calcium and Magnesium. 14. 70 %—Percoll fraction: 70 % SIP plus 30 % PBS, without Calcium and Magnesium (see Note 7).
3
Methods Keep biological material on ice unless otherwise specified. The stated volumes are calculated for the whole CNS, i.e. brain plus spinal cord of one mouse. Cell isolation from separated CNS parts is possible with reduced volumes. If further culturing of the cells is required, the isolation should be performed under sterile conditions, e.g. with sterile instruments, suppliers, solutions and under a laminar flow cabinet (see Note 8).
3.1 CNS Tissue Isolation and Dissociation
1. Deeply anesthetize mouse, make sure there is no pain reflex (see Note 9). 2. Spray mouse with ethanol, cut the skin, and open thoracic cavity. Make a small cut in the right atrium of the heart and insert the perfusion needle 0.9 40 mm into the left cardiac ventricle (see Note 10). 3. Run 40–50 ml of the perfusion solution for about 3–5 min through cannula with low drip until the effluent solution from the right atrium doesn’t consist of blood, i.e. become fully transparent (see Note 11).
Cell Isolation from CNS
4. Decapitate mouse, cut the skin, and isolate the brain out of the skull into a petri dish. Cover isolated brain with 3 ml PBS/FCS (see Note 12). 5. From the mouse body, cut the back skin longwise from the cranial end until the tail basis. Cut the ribs and separate the spinal column down to lumbar region. 6. Fill up 10 ml syringe with 1.2 40 mm needle with PBS/FCS. Fix the spinal column with forceps and insert the needle into the spinal canal at the caudal end. Flush out the spinal cord with PBS/FCS into petri dish (see Note 13). 7. Combine spinal cord and brain in one petri dish, if cell isolation is desired from the whole CNS. 8. Cut CNS tissue into small pieces (about 1 mm2) by using a razor blade and collect everything in a 15 ml Falcon tube. 9. Centrifuge at 300 g, 5 min, 4 C. Remove the supernatant. 10. Resuspend in 1 ml digestion solution by using vortexer and incubate for 30 min, 37 C in water bath. 11. Stop the reaction by placing Falcon tube on ice and adding 3 ml PBS/FCS. 12. Homogenize digested tissue pieces by using the 5 ml syringe with 1.2 40 mm needle. Intensive collection and removal for about 3–5 times is necessary. 13. Add 8 ml PBS/FCS and centrifuge at 300 g, 5 min, 4 C. Remove the supernatant (see Note 14). 14. Resuspend the pellet in 4 ml 70 %—Percoll fraction by using vortexer and collect in 5 ml syringe with 0.9 70 mm needle. 3.2 CNS Infiltrating Cell Purification
1. Add 4 ml 30 %—Percoll fraction into a new 15 ml Falcon tube and carefully underlay with 4 ml 37 %—Percoll fraction by using 5 ml syringe with long 0.9 70 mm needle. Avoid air bubbles and mixing fractions due to turbulence (see Note 15). 2. Carefully underlay 4 ml of 70 %—Percoll fraction containing digested CNS into the Falcon tube from step 1 (already containing the other two Percoll fractions). Again, use the syringe with 0.9 70 mm needle like in step before. Avoid air bubbles and residual tissue clumps (see Note 16). 3. Perform Percoll gradient separation by centrifugation for 40 min at 500 g, RT with the lowest acceleration settings and without brake. After centrifugation the tube should contain a viscose myelin-enriched layer on top. The CNS infiltrating cells and microglia should be visible as an opaque white ring at the interphase of middle 37 % (red) and lower 70 % (white) Percoll fractions. 4. Carefully remove the upper myelin-enriched layer (see Note 17).
Ilgiz A. Mufazalov and Ari Waisman
5. Carefully harvest cells from the interphase described above and transfer into new 15 ml Falcon tube (see Note 18). 6. Add 10 ml PBS/FCS, vortex, and centrifuge at 300 g, 5 min, 4 C (see Note 19). 7. Resuspend cells in PBS/FCS and proceed for further analysis.
4
Notes 1. Using syringes with Luer lock are preferential for safety reasons. 2. Isofluran can be exchanged with other anesthesia. Follow the institutional biohazard regulations. 3. Other isotonic solutions can be used instead of NaCl, for example 1PBS without Calcium and Magnesium. 4. Using HBSS with Phenol Red will help to better visualize different Percoll fractions. 5. Make aliquots of 2 mg/ml DNase I and store at 20 C. 6. Concentration is represented here as volume/volume parts, i.e. take nine parts of Percoll and one part of 10PBS to prepare SIP. 7. Brief calculation for infiltrating cell isolation from 1 mouse CNS (brain plus spinal cord): 5.5 ml SIP ¼ 4.95 ml Percoll + 0.55 ml 10PBS 4 ml 30 %—Percoll ¼ 1.20 ml SIP + 2.80 ml 1PBS 4 ml 37 %—Percoll ¼ 1.48 ml SIP + 2.52 ml 1HBSS 4 ml 70 %—Percoll ¼ 2.80 ml SIP + 1.20 ml 1PBS Prepare different Percoll gradient fractions shortly before usage. Cold Percoll could result in forming cell aggregates; therefore, solutions should be at RT. 8. Working in a team, especially when handling large sample numbers, significantly decreases the time of cell isolation, which is crucial for yield and quality. 9. To check pain reflex pinch the skin with the forceps. If there is a reaction perform longer anesthesia. Important! Deep anesthesia should still keep the heart beating, which is necessary for successful cardiac perfusion. Cervical dislocation is not acceptable as it may disrupt blood vessels and disturb CNS perfusion. 10. The notched right atrium will allow perfusion solution to go out of circulation. 11. Alternatively, instead of i.v. perfusion system, a perfusion pump or manual syringe perfusion can be used. Follow the color change of the liver from dark red to light brown as indicator of successful perfusion.
Cell Isolation from CNS
12. A fully white brain with nonvisible blood vessels is a good indication of successful perfusion. Avoid any residual blood spillover from surrounding tissues. If such blood contamination occurred, wash CNS tissue with high-volume PBS/FCS. In case some parts of the brain are desired for further histological analysis be careful with the cutting and removal of cranium bones. 13. The needle diameter is just slightly below the internal spinal canal diameter. Therefore, tight junction should prevent retrograde spinal cord flushing. If the resistance during flushing is too strong make sure the spinal column is straightened. Once the spinal cord is flushed out, repeat the procedure, now from the cranial end. Avoid any residual blood contamination from surrounded tissues. If blood contamination occurs, wash with high-volume PBS/FCS. 14. In that stage the pellet is loose, be careful with supernatant removal. 15. Filling up the syringe with 5 ml 37 %—Percoll solution and release only 4 ml will help to avoid air bubbles. A clear separation line should be visible at the 30–37 % junction. 16. Be careful when releasing the residual volume from the syringe, this is the most critical step in air bubble generation. 17. Also remove the white ring on top of red fraction if that have had appeared. 18. Collecting cells with 1 ml pipet tips is useful for this purpose. Usually 2 ml is enough for proper cell collection. 19. Proper mixing is necessary for washing out the residual Percoll solution.
Acknowledgement We are thankful to Dr. Tommy Regen for useful discussions and careful correction of the manuscript and to Sonja Lacher for providing FACS plots surface staining. References 1. Wong AD, Ye M, Levy AF, Rothstein JD, Bergles DE, Searson PC (2013) The blood-brain barrier: an engineering perspective. Front Neuroeng 6:7 2. Milo R, Miller A (2014) Revised diagnostic criteria of multiple sclerosis. Autoimmun Rev 13:518–524 3. Pierson E, Simmons SB, Castelli L, Goverman JM (2012) Mechanisms regulating regional localization of inflammation during CNS autoimmunity. Immunol Rev 248:205–215
4. Muller DM, Pender MP, Greer JM (2000) A neuropathological analysis of experimental autoimmune encephalomyelitis with predominant brain stem and cerebellar involvement and differences between active and passive induction. Acta Neuropathol 100:174–182 5. Cardona AE, Huang D, Sasse ME, Ransohoff RM (2006) Isolation of murine microglial cells for RNA analysis or flow cytometry. Nat Protoc 1:1947–1951
Isolation of Central Nervous System (CNS) Infiltrating Cells Ilgiz A. Mufazalov and Ari Waisman Abstract Leukocyte infiltration of the central nervous system (CNS) occurs under certain pathogenic conditions and most often results in severe disorders. Therefore, the isolation and analysis of such infiltrating cell populations is necessary for elucidating the underlying pathogenic mechanisms. Here we describe a simple and straightforward protocol for cell isolation from the inflamed CNS, which combines mechanical dissociation and enzymatic degradation of the tissue. Additionally, purification by Percoll gradient centrifugation provides a great yield of the infiltrating material. The isolated cells can be further used for downstream applications such as cell sorting, cellular or molecular analysis. Keywords: CNS, EAE, Multiple sclerosis, Percoll, T cells, Blood–brain barrier, Protocol
1
Introduction The integrity of the blood–brain barrier (BBB) determines proper trafficking of nutrients and (cellular) mediators into and from the CNS parenchyma, and therefore represents a key feature of the healthy individual. However, under certain circumstances disruption of the BBB leads to severe dysfunctions. Thus, BBB breakdown has been associated, among others, with Alzheimer’s disease, Parkinson’s disease, brain infections, stroke, epilepsy, and multiple sclerosis (MS) (1). MS is a common neurodegenerative disorder that affects mainly Caucasians in high-income countries and the number of people suffering from MS has increased in the last years and reached about 2.5 millions all over the world (2). The disease progression is characterized by the presence of multiple inflammatory and demyelinating lesions within the brain and spinal cord, which result in axonal damage and consequently in clinical symptoms such as sensory and motor impairment, ataxia, spasticity, fatigue, and cognitive impairment (3). MS is believed to be of autoimmune nature and has a clear inflammatory component. Despite intensive investigations during past decades the exact mechanisms of disease onset and progression still remain unknown. To study MS in more detail an animal model termed experimental autoimmune encephalomyelitis (EAE) has been established. EAE mimics many of the MS features in rodents and, despite all its
Ilgiz A. Mufazalov and Ari Waisman
limitations, remains the best tool to study the disease in vivo. Like in MS, EAE progression is also characterized by inflammation and accumulation of infiltrating peripheral cells within the CNS. Interestingly, EAE clinical symptoms can be different according to the main site of inflammation being the brain or spinal cord (4). Therefore, CNS infiltrating cell isolation is a crucial and indispensible step for understanding the mechanisms of MS/EAE pathogenesis. Here we describe a detailed protocol for the isolation of CNS infiltrating cells from animals suffering from mouse models of the aforementioned disorders with CNS associated immune responses, such as EAE. An essential basis for the protocol is the cardiac perfusion of the animals with normal saline solution to avoid possible peripheral blood cell contamination of the CNS samples. In the second step a combination of mechanical dissociation and enzymatic degradation of the extracellular matrix proteins is implemented to achieve a single cell suspension. Further purification with the colloid Percoll is necessary to enrich for CNS infiltrating cells. Pecoll is not toxic to the cells under these conditions and does not interfere with many laboratory applications, such as electronic counting instruments or fluorescent activated cell sorting (FACS). Therefore, isolated CNS infiltrating cells may be used for further analysis and characterization. Importantly, the protocol is not designed to isolate only mononuclear CNS infiltrating cells but rather attempts to purify these cells from debris, extracellular components, and most importantly the myelin. The critical step of a discontinuous three-layer Percoll gradient leads to enrichment of leukocytes as well as CNS resident cell types (such as microglia (5)) in the interphases. Beneficially, these CNS resident cell types may be used as internal control for infiltrating cell quantification. Therefore, additional purification procedures are required to isolate specific CNS infiltrating cell subsets. Total numbers of CNS infiltrating cells are strongly correlated with EAE severity and the stage of disease progression. For example, to yield the highest amounts of T cells from the diseased CNS the isolation should be performed at the peak of disease with pooled brain and spinal cord material. Using the here described method, isolation of CNS (infiltrating) cells from mice with fully paralyzed hind limbs (score 3.5), partially (score 4) or fully (score 4.5) paralyzed front limbs routinely yields 1.5–3.5 million cells, including about 0.5 million CD90 positive T cells (Fig. 1a). Importantly, the isolated cells may be further used for downstream application, e.g. the flow cytometric analysis to distinguish infiltrated lymphocytes (CD45+CD11b), macrophages (CD45highCD11b+) and microglia (CD45lowCD11b+) (Fig. 1b). Moreover, different effector T cell subsets maybe be identified and characterized based on their characteristic cytokine expression (Fig. 1c).
Cell Isolation from CNS
Fig. 1 Isolation of CNS infiltrating cells from C57Bl/6 mice with EAE. (a) Absolute numbers of live and CD90 positive cells. Data represents CNS isolated material from 19 EAE diseased mice, with clinical scores ranging from 3.5 to 4.5, from 7 independent experiments. (b) Surface markers expression profile of CNS infiltrating lymphocytes (CD45+CD11b), macrophages and microglia represents greater amounts of inflammatory cells in EAE diseased mouse (score 4) compare to nondiseased mouse (score 0). (c) Cytokine expression profile of CNS infiltrated CD4+CD40L+ T cells isolated from a mouse with clinical score 4. Cells were stimulated ex vivo in the presence of MOG peptide and Brefeldin A for 6 h
2
Materials Prepare all equipment and materials in advance, as the time is critical for cell survival during the isolation procedure. Only digestion solution and Percoll fractions should be prepared shortly before usage.
2.1
Equipment
1. Surgical instruments: scissors, forceps, i.v. perfusion system, razor blades. 2. Suppliers: 100 mm petri dishes, 15 ml Falcon tubes, 5 ml and 10 ml medical syringes, hypodermic needles (BD): 0.9 40 mm 20 G “yellow,” 1.2 40 mm 18 G “red,” 0.9 70 mm 20 G “long yellow” (see Note 1). 3. Equipment: vortexer, centrifuge with swing-out bucket rotor, water bath.
2.2
Reagents
1. Mouse anesthesia: Isofluran (see Note 2). 2. 70 % Ethanol.
Ilgiz A. Mufazalov and Ari Waisman
3. Perfusion solution: Normal saline (0.9 % w/v NaCl) (see Note 3). 4. Phosphate buffered saline (PBS), without Calcium and Magnesium. 5. PBS, with Calcium and Magnesium. 6. 10PBS, without Calcium and Magnesium. 7. Hanks’ Balanced Salt Solution (HBSS), without Calcium and Magnesium (see Note 4). 8. Fetal calf serum (FCS). 9. Cell isolation/storage buffer (PBS/FCS): PBS supplemented with 2 % FCS. 10. Digestion solution: PBS with Calcium and Magnesium, 1.5 mg/ml Collagenase II, 50 μg/ml DNase I (see Note 5). 11. Stock Isotonic Percoll (SIP) solution: 90 % Percoll plus 10 % 10PBS, without Calcium and Magnesium (see Note 6). 12. 30 %—Percoll fraction: 30 % SIP plus 70 % PBS, without Calcium and Magnesium. 13. 37 %—Percoll fraction: 37 % SIP plus 63 % HBSS, without Calcium and Magnesium. 14. 70 %—Percoll fraction: 70 % SIP plus 30 % PBS, without Calcium and Magnesium (see Note 7).
3
Methods Keep biological material on ice unless otherwise specified. The stated volumes are calculated for the whole CNS, i.e. brain plus spinal cord of one mouse. Cell isolation from separated CNS parts is possible with reduced volumes. If further culturing of the cells is required, the isolation should be performed under sterile conditions, e.g. with sterile instruments, suppliers, solutions and under a laminar flow cabinet (see Note 8).
3.1 CNS Tissue Isolation and Dissociation
1. Deeply anesthetize mouse, make sure there is no pain reflex (see Note 9). 2. Spray mouse with ethanol, cut the skin, and open thoracic cavity. Make a small cut in the right atrium of the heart and insert the perfusion needle 0.9 40 mm into the left cardiac ventricle (see Note 10). 3. Run 40–50 ml of the perfusion solution for about 3–5 min through cannula with low drip until the effluent solution from the right atrium doesn’t consist of blood, i.e. become fully transparent (see Note 11).
Cell Isolation from CNS
4. Decapitate mouse, cut the skin, and isolate the brain out of the skull into a petri dish. Cover isolated brain with 3 ml PBS/FCS (see Note 12). 5. From the mouse body, cut the back skin longwise from the cranial end until the tail basis. Cut the ribs and separate the spinal column down to lumbar region. 6. Fill up 10 ml syringe with 1.2 40 mm needle with PBS/FCS. Fix the spinal column with forceps and insert the needle into the spinal canal at the caudal end. Flush out the spinal cord with PBS/FCS into petri dish (see Note 13). 7. Combine spinal cord and brain in one petri dish, if cell isolation is desired from the whole CNS. 8. Cut CNS tissue into small pieces (about 1 mm2) by using a razor blade and collect everything in a 15 ml Falcon tube. 9. Centrifuge at 300 g, 5 min, 4 C. Remove the supernatant. 10. Resuspend in 1 ml digestion solution by using vortexer and incubate for 30 min, 37 C in water bath. 11. Stop the reaction by placing Falcon tube on ice and adding 3 ml PBS/FCS. 12. Homogenize digested tissue pieces by using the 5 ml syringe with 1.2 40 mm needle. Intensive collection and removal for about 3–5 times is necessary. 13. Add 8 ml PBS/FCS and centrifuge at 300 g, 5 min, 4 C. Remove the supernatant (see Note 14). 14. Resuspend the pellet in 4 ml 70 %—Percoll fraction by using vortexer and collect in 5 ml syringe with 0.9 70 mm needle. 3.2 CNS Infiltrating Cell Purification
1. Add 4 ml 30 %—Percoll fraction into a new 15 ml Falcon tube and carefully underlay with 4 ml 37 %—Percoll fraction by using 5 ml syringe with long 0.9 70 mm needle. Avoid air bubbles and mixing fractions due to turbulence (see Note 15). 2. Carefully underlay 4 ml of 70 %—Percoll fraction containing digested CNS into the Falcon tube from step 1 (already containing the other two Percoll fractions). Again, use the syringe with 0.9 70 mm needle like in step before. Avoid air bubbles and residual tissue clumps (see Note 16). 3. Perform Percoll gradient separation by centrifugation for 40 min at 500 g, RT with the lowest acceleration settings and without brake. After centrifugation the tube should contain a viscose myelin-enriched layer on top. The CNS infiltrating cells and microglia should be visible as an opaque white ring at the interphase of middle 37 % (red) and lower 70 % (white) Percoll fractions. 4. Carefully remove the upper myelin-enriched layer (see Note 17).
Ilgiz A. Mufazalov and Ari Waisman
5. Carefully harvest cells from the interphase described above and transfer into new 15 ml Falcon tube (see Note 18). 6. Add 10 ml PBS/FCS, vortex, and centrifuge at 300 g, 5 min, 4 C (see Note 19). 7. Resuspend cells in PBS/FCS and proceed for further analysis.
4
Notes 1. Using syringes with Luer lock are preferential for safety reasons. 2. Isofluran can be exchanged with other anesthesia. Follow the institutional biohazard regulations. 3. Other isotonic solutions can be used instead of NaCl, for example 1PBS without Calcium and Magnesium. 4. Using HBSS with Phenol Red will help to better visualize different Percoll fractions. 5. Make aliquots of 2 mg/ml DNase I and store at 20 C. 6. Concentration is represented here as volume/volume parts, i.e. take nine parts of Percoll and one part of 10PBS to prepare SIP. 7. Brief calculation for infiltrating cell isolation from 1 mouse CNS (brain plus spinal cord): 5.5 ml SIP ¼ 4.95 ml Percoll + 0.55 ml 10PBS 4 ml 30 %—Percoll ¼ 1.20 ml SIP + 2.80 ml 1PBS 4 ml 37 %—Percoll ¼ 1.48 ml SIP + 2.52 ml 1HBSS 4 ml 70 %—Percoll ¼ 2.80 ml SIP + 1.20 ml 1PBS Prepare different Percoll gradient fractions shortly before usage. Cold Percoll could result in forming cell aggregates; therefore, solutions should be at RT. 8. Working in a team, especially when handling large sample numbers, significantly decreases the time of cell isolation, which is crucial for yield and quality. 9. To check pain reflex pinch the skin with the forceps. If there is a reaction perform longer anesthesia. Important! Deep anesthesia should still keep the heart beating, which is necessary for successful cardiac perfusion. Cervical dislocation is not acceptable as it may disrupt blood vessels and disturb CNS perfusion. 10. The notched right atrium will allow perfusion solution to go out of circulation. 11. Alternatively, instead of i.v. perfusion system, a perfusion pump or manual syringe perfusion can be used. Follow the color change of the liver from dark red to light brown as indicator of successful perfusion.
Cell Isolation from CNS
12. A fully white brain with nonvisible blood vessels is a good indication of successful perfusion. Avoid any residual blood spillover from surrounding tissues. If such blood contamination occurred, wash CNS tissue with high-volume PBS/FCS. In case some parts of the brain are desired for further histological analysis be careful with the cutting and removal of cranium bones. 13. The needle diameter is just slightly below the internal spinal canal diameter. Therefore, tight junction should prevent retrograde spinal cord flushing. If the resistance during flushing is too strong make sure the spinal column is straightened. Once the spinal cord is flushed out, repeat the procedure, now from the cranial end. Avoid any residual blood contamination from surrounded tissues. If blood contamination occurs, wash with high-volume PBS/FCS. 14. In that stage the pellet is loose, be careful with supernatant removal. 15. Filling up the syringe with 5 ml 37 %—Percoll solution and release only 4 ml will help to avoid air bubbles. A clear separation line should be visible at the 30–37 % junction. 16. Be careful when releasing the residual volume from the syringe, this is the most critical step in air bubble generation. 17. Also remove the white ring on top of red fraction if that have had appeared. 18. Collecting cells with 1 ml pipet tips is useful for this purpose. Usually 2 ml is enough for proper cell collection. 19. Proper mixing is necessary for washing out the residual Percoll solution.
Acknowledgement We are thankful to Dr. Tommy Regen for useful discussions and careful correction of the manuscript and to Sonja Lacher for providing FACS plots surface staining. References 1. Wong AD, Ye M, Levy AF, Rothstein JD, Bergles DE, Searson PC (2013) The blood-brain barrier: an engineering perspective. Front Neuroeng 6:7 2. Milo R, Miller A (2014) Revised diagnostic criteria of multiple sclerosis. Autoimmun Rev 13:518–524 3. Pierson E, Simmons SB, Castelli L, Goverman JM (2012) Mechanisms regulating regional localization of inflammation during CNS autoimmunity. Immunol Rev 248:205–215
4. Muller DM, Pender MP, Greer JM (2000) A neuropathological analysis of experimental autoimmune encephalomyelitis with predominant brain stem and cerebellar involvement and differences between active and passive induction. Acta Neuropathol 100:174–182 5. Cardona AE, Huang D, Sasse ME, Ransohoff RM (2006) Isolation of murine microglial cells for RNA analysis or flow cytometry. Nat Protoc 1:1947–1951
