This research was supported by National Natural Science Foundation of China grant (31570921 to ZJ, 81571533 to LS), Shanghai Municipal Commission of Health, and Family Planning (201540206 to ZJ), Ruijin Hospital North research grant (2017ZX01 to ZJ).

All methods described here have been approved by the animal committee of the School of Basic Medical Sciences, Shanghai Jiao Tong University.
1. Preparation of the materials
<ol>
	<li>Use the MEVGWYRSPFSRVVHLYRNGK sequence of MOG35&#8211;55 to obtain the lyophilized peptide from commercial sources. Ensure that the purity of the peptide is &#62;95%. Prepare 10 mg/mL MOG stock solution in phosphate-buffered saline (PBS) and store at -20 &#176;C.</li>
	<li>Prepare a 4 mg/mL stock solution of M. tuberculosis H37 Ra by putting one 100 mg tube of M. tuberculosis H37 Ra into 25 mL of Complete Freund&#39;s Adjuvant (CFA) and mixing. Store the stock solution at -20 &#176;C.</li>
	<li>Prepare 1 ng/&#956;L Pertussis Toxin Working Solution (PTX) by adding 50 &#956;g of PTX into 50 mL of PBS. Store the working solution at -20 &#176;C.</li>
	<li>Store all antibodies (i.e., FITC anti-mouse CD3, PE/Cy7 anti-mouse CD4, PerCP/Cy5.5 anti-mouse CD11b, Alexa Fluor700 anti-mouse CD45.2, PE anti-mouse IL-17A, and APC anti-mouse IFN-&#947;) at 4 &#176;C.</li>
	<li>Make the Flow Cytometry Staining (FCS) Buffer by adding 2 mM ethylene diamine tetraacetic acid (EDTA) and 1% fetal bovine serum (FBS) into 500 mL of PBS.</li>
</ol>
2. Housing of C57BL/6 mice
<ol>
	<li>Use female C57BL/6 mice at 8&#8211;12 weeks of age.</li>
	<li>Acclimate C57BL/6J mice for at least 7 days prior to the injection.</li>
	<li>House mice in an animal facility under pathogen-free conditions at constant temperature and humidity in a 12 h light/dark cycle and provide free access to water and standard pellet food.</li>
</ol>
3. Immunization of C57BL/6 mice
<ol>
	<li>Leave all stock solutions for 15 min at room temperature (RT) to ensure complete rehydration.</li>
	<li>Dilute 300 &#956;L of MOG-peptide stock solution with 700 &#956;L of PBS for preparing 3 mg/mL work solution.</li>
	<li>Put 1 mL of M. tuberculosis H37 Ra stock solution and 1 mL of MOG35&#8211;55 peptides working solution into separate 10 mL syringes, then use a four-way stop cock to emulsify for at least 10 min. Ensure complete emulsification before injection.</li>
	<li>Anesthetize mice at the peak of EAE with an intraperitonial injection of 1% sodium pentobarbital (50 mg/kg).</li>
	<li>With a 1 mL syringe, subcutaneously inject mice with 100 &#956;L of a MOG 35&#8211;55/CFA emulsion (300 &#181;g/200 &#956;L) at two sites, both at the back near the neck. Subcutaneously inject control mice with 200 &#956;L of PBS.</li>
	<li>On the same day (day 0) and on day 2 post immunization (PI), Intraperitoneally inject mice with 200 &#956;L of 1 ng/&#956;L PTX working solution. Intraperitoneally&#160;inject control mice with 200 &#956;L of PBS.</li>
	<li>Transfer the mice to their home cage with a warming pad.</li>
	<li>Examine and grade all mice every day after the injection in a blinded manner for the neurological signs shown in Table 111,13. Euthanize the animals if the scores are worse than grade 4.</li>
	<li>Record the weight changes during the disease course. This is a valuable additional measure for disease activity in the EAE model11,13.</li>
	<li>Add the first day of clinical signs for individual mice and divide by the number of mice in the group; the result is the onset. Add the first day of the maximum EAE score for individual mice and divide by the number of mice in the group; the result is the peak.</li>
</ol>
4. Single-cell suspension preparation from brain
<ol>
	<li>Dilute density gradient medium in 9:1 ratio with PBS in a 15 mL conical tube to yield a final 100% solution.</li>
	<li>Anesthetize mice at the peak of EAE with an intraperitonial injection of 1% sodium pentobarbital (50 mg/kg) and perfuse intracardially with 20 mL of sterile ice-cold PBS. Achieve this by slowly and steadily injecting PBS into the left ventricle of the heart using a 20 mL syringe and opening the right atrium.</li>
	<li>Cut the cranium carefully from the nose to the neck, then remove the brain from the cranial box into 10 mL of RPMI in 50 mL conical tubes. Mix well to remove adherent red blood cells. Then remove the medium by aspiration and add 10 mL of RPMI.</li>
	<li>Place the brains and medium in a 100 mm dish. Finely chop with a razor blade.</li>
	<li>Transfer 6 mL from the dish to an ice-cold 7 mL sintered glass homogenizer with a clean pipette. Avoid leaving large quantities of tissue in the pipette. A small amount is unavoidable.</li>
	<li>Grind the brain using the &#34;loose&#34; plunger of the pestle first, then use the &#34;tight&#34; plunger until the suspension is homogeneous, and pour into a prechilled 15 mL conical tube and keep on ice.</li>
	<li>After all the samples are homogenized, estimate the volume. Adjust the volume with RPMI to 7 mL. Then place 3 mL of ice-cold 100% basement membrane matrix in a chilled 15 mL conical centrifuge tube and add 7 mL of the brain homogenate to yield a final 30% density gradient medium. Mix by inversion a couple of times. Do not vortex.</li>
	<li>To ensure a sharp interface, carefully and slowly add 1 mL of 70% underlay density gradient medium in RPMI with a 3 mL pipette.</li>
	<li>Centrifuge at 800 x g for only 20 min at 4 &#176;C. Set the acceleration to 1 and deceleration to 0. After centrifugation, aspirate almost all of the top phase, being careful to completely remove the myelin at the top (Figure 1).</li>
	<li>Remove the interface into a new 15 centrifuge tube. Adjust the volume to 10 mL with RPMI.</li>
	<li>Centrifuge at 500 x g for 10 min. After centrifugation, aspirate the supernatant. Resuspend the pellet in ~200 &#956;L of flow cytometry staining (FSC) buffer. The pellets are then ready to stain for FACS.</li>
</ol>
5. Flow cytometric analysis of single cells from brain
<ol>
	<li>Use a hemocytometer and microscope to count the cells. Add 10 &#956;L of the cells to 10 &#956;L of trypan blue, mix well, and place 10 &#956;L on a hemocytometer to count the cells. Then calculate the number of live cells per microliter under an inverted microscope (e.g., Olympus Inverted microscope).</li>
	<li>Aliquot approximately 2 x 106 of cells in RPMI into a single well of a 96 well plate.</li>
	<li>Add 500x cell stimulation cocktail plus protein transport inhibitors to the wells.</li>
	<li>Incubate the plate in the incubator at 37 &#176;C for 4 h.</li>
	<li>Centrifuge the cells at 400 x g for 5 min at RT. Discard the supernatant and resuspend the cells in 100 &#956;L of FCS Buffer.</li>
	<li>Preincubate the cells with anti-mouse CD16/CD32 Fc block (1:33) for 10 min at 4 &#176;C before staining to block nonspecific Fc-mediated interactions.</li>
	<li>Stain cell surface markers without washing. Add anti-mouse CD45.2 (1:200), anti-mouse CD11b (1:200), anti-mouse CD3 (1:200), and anti-mouse CD4 (1:200) antibodies. 
	NOTE: To determine positive and negative gates, a fluorescence minus one (FMO) for each color and an isotype control antibody should be stained.</li>
	<li>Incubate the plate for at least 30 min at 4 &#176;C or on ice. Protect from light.</li>
	<li>Wash the cells by adding FCS Buffer. Use 200 &#956;L/well for microtiter plates. Centrifuge at 400 x g for 5 min at RT. Discard the supernatant.</li>
	<li>Add 200 &#956;L of intracellular (IC) fixation buffer to each well to fix the cells. Ensure the cells are fully resuspended in the solution.</li>
	<li>Incubate 30&#8211;60 min at RT. Protect from light.</li>
	<li>Centrifuge the samples at 400 x g at RT for 5 min. Discard the supernatant.</li>
	<li>Add 200 &#956;L of 1x permeabilization buffer to each well and centrifuge the samples at 400 x g at RT for 5 min. Discard the supernatant.</li>
	<li>Resuspend the pellet in residual volume and adjust volume to about 100 &#956;L with 1x permeabilization buffer.</li>
	<li>Add anti-mouse IL-17A (1:200) and anti-mouse IFN-g (1:200) antibodies for detection of intracellular antigens to cells.</li>
	<li>Incubate for at least 30 min at 4 &#176;C. Protect from light.</li>
	<li>Add 100 &#956;L of 1x permeabilization buffer to each well and centrifuge the samples at 400 x g at RT for 5 min. Discard the supernatant.</li>
	<li>Resuspend the stained cells in 100 &#956;L of flow cytometry staining buffer.</li>
	<li>Analyze by flow cytometry. 
	NOTE: The laser and compensation settings on the flow cytometer are adjusted, the samples are placed onto the cytometer, and all events are recorded as per the manufacturer&#8217;s recommendations.</li>
</ol>
6. Data analysis
<ol>
	<li>Gate singlets using FSC-A vs. FSC-H and SSC-A vs. SSC-H.</li>
	<li>Gate live cells using FSC-A and SSC-A based on size.</li>
	<li>Next, identify the leukocytes, excluding the monocytes, by gating on CD45+ CD11b- cells.</li>
	<li>Then, identify the CD4 T lymphocytes by gating on CD3+CD4+ cells.</li>
	<li>Lastly, identify the Th1 and Th17 subsets by gating on IFN-&#947;+ cells and IL-17+ cells separately, and determine the positive and negative populations using isotype controls and FMO.</li>
</ol>

<ol>
	<li>Milo, R., Kahana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sclerosis:+geoepidemiology,+genetics+and+the+environment.">Multiple sclerosis: geoepidemiology, genetics and the environment.</a> Autoimmunity Reviews. 9, 387-394 (2010).</li><li>Gold, R., Linington, C., Lassmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+pathogenesis+and+therapy+of+multiple+sclerosis+via+animal+models:+70+years+of+merits+and+culprits+in+experimental+autoimmune+encephalomyelitis+research.">Understanding pathogenesis and therapy of multiple sclerosis via animal models: 70 years of merits and culprits in experimental autoimmune encephalomyelitis research.</a> Brain : A Journal of Neurology. 129, 1953-1971 (2006).</li><li>Simmons, S. B., Pierson, E. R., Lee, S. Y., Goverman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+the+heterogeneity+of+multiple+sclerosis+in+animals.">Modeling the heterogeneity of multiple sclerosis in animals.</a> Trends in Immunology. 34 (8), 410-422 (2013).</li><li>Lassmann, H., Wisniewski, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+relapsing+experimental+allergic+encephalomyelitis:+clinicopathological+comparison+with+multiple+sclerosis.">Chronic relapsing experimental allergic encephalomyelitis: clinicopathological comparison with multiple sclerosis.</a> Archives of Neurology. 36, 490-497 (1979).</li><li>Bernard, C. C., Leydon, J., Mackay, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+necessity+in+the+pathogenesis+of+experimental+autoimmune+encephalomyelitis+in+mice.">T cell necessity in the pathogenesis of experimental autoimmune encephalomyelitis in mice.</a> European Journal of Immunology. 6, 655-660 (1976).</li><li>Yasuda, T., Tsumita, T., Nagai, Y., Mitsuzawa, E., Ohtani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+allergic+encephalomyelitis+(EAE)+in+mice.+I.+Induction+of+EAE+with+mouse+spinal+cord+homogenate+and+myelin+basic+protein.">Experimental allergic encephalomyelitis (EAE) in mice. I. Induction of EAE with mouse spinal cord homogenate and myelin basic protein.</a> Japanese Journal of Experimental Medicine. 45, 423-427 (1975).</li><li>Mendel, I., Kerlero de Rosbo, N., Ben-Nun, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+myelin+oligodendrocyte+glycoprotein+peptide+induces+typical+chronic+experimental+autoimmune+encephalomyelitis+in+H-2b+mice:+fine+specificity+and+T+cell+receptor+V+beta+expression+of+encephalitogenic+T+cells.">A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H-2b mice: fine specificity and T cell receptor V beta expression of encephalitogenic T cells.</a> European Journal of Immunology. 25, 1951-1959 (1995).</li><li>Bramow, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demyelination+versus+remyelination+in+progressive+multiple+sclerosis.">Demyelination versus remyelination in progressive multiple sclerosis.</a> Brain. 133, 2983-2998 (2010).</li><li>Sospedra, M., Martin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+of+multiple+sclerosis.">Immunology of multiple sclerosis.</a> Annual Reviews in Immunology. 23, 683-747 (2005).</li><li>McGinley, A. M., Edwards, S. C., Raverdeau, M., Mills, K. H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th17+cells,+gammadelta+T+cells+and+their+interplay+in+EAE+and+multiple+sclerosis.">Th17 cells, gammadelta T cells and their interplay in EAE and multiple sclerosis.</a> Journal of Autoimmunity. 20 (14), 3394 (2018).</li><li>Ji, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiamine+deficiency+promotes+T+cell+infiltration+in+experimental+autoimmune+encephalomyelitis:+the+involvement+of+CCL2.">Thiamine deficiency promotes T cell infiltration in experimental autoimmune encephalomyelitis: the involvement of CCL2.</a> Journal of Immunology. 193, 2157-2167 (2014).</li><li>Manglani, M., Gossa, S., McGavern, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+Isolation+from+Brain,+Spinal+Cord,+and+Meninges+for+Flow+Cytometric+Analysis.">Leukocyte Isolation from Brain, Spinal Cord, and Meninges for Flow Cytometric Analysis.</a> Current Protocols in Immunology. 121, 44 (2018).</li><li>Ji, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+promotes+EAE+through+IL-6+and+MCP-1-mediated+T+cells+infiltration.">Obesity promotes EAE through IL-6 and MCP-1-mediated T cells infiltration.</a> Frontiers in Immunology. 10, 1881 (2019).</li><li>Reiseter, B. S., Miller, G. T., Happ, M. P., Kasaian, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+murine+experimental+autoimmune+encephalomyelitis+with+a+myelin+basic+protein+peptide+analog+alters+the+cellular+composition+of+leukocytes+infiltrating+the+cerebrospinal+fluid.">Treatment of murine experimental autoimmune encephalomyelitis with a myelin basic protein peptide analog alters the cellular composition of leukocytes infiltrating the cerebrospinal fluid.</a> Journal of Neuroimmunology. 91, 156-170 (1998).</li><li>Bittner, S., Afzali, A. M., Wiendl, H., Meuth, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+oligodendrocyte+glycoprotein+(MOG35-55)+induced+experimental+autoimmune+encephalomyelitis+(EAE)+in+C57BL/6+mice.">Myelin oligodendrocyte glycoprotein (MOG35-55) induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice.</a> Journal of Visualized Experiments. (86), e51275 (2014).</li><li>Miller, S. D., Karpus, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+autoimmune+encephalomyelitis+in+the+mouse.">Experimental autoimmune encephalomyelitis in the mouse.</a> Current Protocols in Immunology. , 11 (2007).</li><li>Tietz, S. M., Engelhardt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+Impairment+of+the+Endothelial+and+Glial+Barriers+of+the+Neurovascular+Unit+during+Experimental+Autoimmune+Encephalomyelitis+In+Vivo.">Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo.</a> Journal of Visualized Experiments. (145), e59249 (2019).</li></ol>

The authors have no conflicts of interest to declare.

This study presents a protocol to induce and monitor EAE using MOG35-55 in C57BL/6 mice, which are considered a typical neuroimmunological experimental animal model of MS. EAE can be induced varying the mice strains or the type of protein used for induction according to the purpose of the study. For example, using PLP139&#8211;151 peptide in SJL mice can induce a relapsing-remitting EAE disease course that is especially well-suited for assessing therapeutic effects on relapses15. The experimental ...

As a chronic demyelinating disease of the CNS, MS affects about 2.5 million people worldwide and lacks curative treatments1. It is also considered an autoimmune disease, in which myelin antigen specific T lymphocytes initiate an inflammatory reaction and lead to demyelination and axonal injury in the CNS2. Experimental autoimmune encephalomyelitis (EAE) has been widely used to investigate pathogenic mechanisms of MS as a classic autoimmune demyelination disease model in CNS3. There are two ways to induce EAE: one is to induce EAE actively by immunizing animals with myelin components, another is adoptive transfer by transferring encephalitogenic T cells into receptor2,4,5. The susceptibilities to EAE are different in different animal strains6. In C57BL/6 mice, myelin oligodendrocyte glycoprotein (MOG) 35&#8211;55 challenge induces a monophasic disease with extensive demyelination and inflammation in the CNS, which is frequently used in experiments with gene-targeted mice7.
The generation of myelin-specific reactive T cells is required for the occurrence and development of disease in EAE and is an immunological sign of both EAE and MS. Activated autoreactive T lymphocytes cross the blood brain barrier (BBB) into the healthy CNS and initiate EAE disease. When MOG 35&#8211;55 Ag is encountered, these T lymphocytes induce inflammation and the recruitment of effector cells into the CNS, resulting in demyelination and axon destruction8,9. In the EAE model, there is ample evidence that neuroantigen-specific CD4+ T cells can initiate and sustain neuroinflammation and pathology3,10. Depending on the major cytokines produced, CD4+ T lymphocytes have been classified into different subsets: Th1 (characterized by the production of interferon-&#947;), Th2 (characterized by the production of interleukin 4), and Th17 (characterized by the production of interleukin 17). It is believed that activation of Th1 and Th17 cells contribute to the induction, maintenance, and regulation of inflammatory demyelination in EAE and MS by secreting effector cytokines IFN-&#947; and IL-17, which are capable of activating macrophages and recruiting neutrophils to the inflammatory sites to accelerate the lesions11.
Because autoreactive T cells cross the BBB into the CNS and induce the development of disease in MS and EAE, it is very important to analyze T cells in the CNS. However, there are very few established protocols for the isolation of lymphocytes from the CNS12. Therefore, a method optimized for isolating mononuclear cells from the brain and analyzing T lymphocytes with markers CD45, CD11b, CD3, CD4, INF-g, and IL-17 for flow cytometry was developed. The method uses MOG35&#8211;55 adjuvant Mycobacterium tuberculosis H37 Ra and Pertussis Toxin Working Solution (PTX) to induce an active immunization model of EAE in mice. Then, mechanical separation and density gradient centrifugation methods are used for the isolation of CNS mononuclear cells. Finally, an optimized flow cytometry gating strategy is used to identify T lymphocytes and subsets from the brain by staining multiple markers.

<table>
	<tbody>
		<tr>
			<td>Alexa Fluor700 anti-mouse CD45.2</td>
			<td>eBioscience</td>
			<td>56-0454-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD16/CD32 Fc block</td>
			<td>BioLegend</td>
			<td>101302</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse IFN-g</td>
			<td>eBioscience</td>
			<td>17-7311-82</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRFortessa X-20</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce homogenizer</td>
			<td>Wheaton</td>
			<td>353107542</td>
			<td></td>
		</tr>
		<tr>
			<td>eBioscience Cell Stimulation Cocktail (plus protein transport inhibitors) (500X)</td>
			<td>eBioscience</td>
			<td>00-4975-03</td>
			<td></td>
		</tr>
		<tr>
			<td>eBioscience Intracellular Fixation &#38; Permeabilization Buffer Set</td>
			<td>eBioscience</td>
			<td>88-8824-00</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD3</td>
			<td>BioLegend</td>
			<td>100203</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Rat IgG2b, &#954; Isotype Ctrl Antibody</td>
			<td>BioLegend</td>
			<td>400605</td>
			<td></td>
		</tr>
		<tr>
			<td>Freund&#39;s Adjuvant Complete (CFA)</td>
			<td>Sigma-Aldrich</td>
			<td>F5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG2a kappa Isotype Control (eBM2a), Alexa Fluor 700, eBioscience</td>
			<td>eBioscience</td>
			<td>56-4724-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycobacterium tuberculosis H37 Ra</td>
			<td>Difco Laboratories</td>
			<td>231141</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse IL-17A</td>
			<td>eBioscience</td>
			<td>12-7177-81</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cy7 anti-mouse CD4</td>
			<td>BioLegend</td>
			<td>100422</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cy7 Rat IgG2b, &#954; Isotype Ctrl Antibody</td>
			<td>BioLegend</td>
			<td>400617</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5 anti-mouse CD11b</td>
			<td>BioLegend</td>
			<td>101228</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5 Rat IgG2b, &#954; Isotype Ctrl Antibody</td>
			<td>BioLegend</td>
			<td>400631</td>
			<td></td>
		</tr>
		<tr>
			<td>pertussis toxin (PTX)</td>
			<td>Sigma-Aldrich</td>
			<td>P-2980</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG1 kappa Isotype Control (eBRG1), APC, eBioscience</td>
			<td>eBioscience</td>
			<td>17-4301-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2a kappa Isotype Control (eBR2a), PE, eBioscience</td>
			<td>eBioscience</td>
			<td>12-4321-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat MOG35&#8211;55 peptides</td>
			<td>Biosynth International</td>
			<td></td>
			<td>MEVGWYRSPFSRVVHLYRNGK</td>
		</tr>
	</tbody>
</table>

flow cytometric analysis of lymphocyte infiltration in central nervous system during experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

After immunization of C57BL/6 mice, all mice were weighed, examined, and graded daily for neurological signs. The representative clinical course of EAE should result in a disease curve as presented in Figure 2A and a change of body weight in the mouse as presented in Figure 2B. C57BL/6 mice immunized with MOG35-55 usually started to develop disease symptoms around day 10&#8211;12 and achieved the peak of disease around day 15&#8211;21 after active immunization (...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis at JoVE.com

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

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JoVE Journal

Immunology and Infection

6.9K Views.  Shanghai Jiao Tong University School of Medicine. This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Video: Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 &#181;L of solution in 1-2 &#181;L droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

Immunogenicity Enhancement by Mycobacterium paratuberculosis in Autoimmune Encephalomyelitis

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in complete Freund&#39;s adjuvant (CFA) containing inactivated Mycobacterium tuberculosis. The antigenic components of the mycobacterium activate dendritic cells to stimulate T-cells to produce cytokines that promote the Th1 response via toll-like receptors. Therefore, the amount and species of mycobacteria present during the antigenic challenge are directly related to the development of EAE. This methods paper presents an alternative protocol to induce EAE in C57BL/6 mice using a modified incomplete Freund&#39;s adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis strain K-10.
M. paratuberculosis, a member of the Mycobacterium avium complex, is the causative agent of Johne&#39;s disease in ruminants and has been identified as a risk factor for several human T-cell-mediated disorders, including multiple sclerosis. Overall, mice immunized with Mycobacterium paratuberculosis showed earlier onset and greater disease severity than mice immunized with CFA containing the strain of M. tuberculosis H37Ra at the same doses of 4 mg/mL. The antigenic determinants of Mycobacterium avium subspecies paratuberculosis (MAP)&#160;strain K-10 were able to induce a strong Th1 cellular response during the effector phase, characterized by significantly higher numbers of T-lymphocytes (CD4+ CD27+), dendritic cells (CD11c+ I-A/I-E+), and monocytes (CD11b+ CD115+) in the spleen compared to mice immunized with CFA. Furthermore, the proliferative T-cell response to the MOG peptide appeared to be highest in M. paratuberculosis-immunized mice. The use of an encephalitogen (e.g., MOG35-55) emulsified in an adjuvant containing M. paratuberculosis in the formulation may be an alternative and validated method to activate dendritic cells for priming myelin epitope-specific CD4+ T-cells during the induction phase of EAE.

Author Spotlight: Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis  

Here, we present an alternative protocol to actively induce experimental autoimmune encephalomyelitis in C57BL/6 mice, using the immunogenic epitope myelin oligodendrocyte glycoprotein (MOG)35-55 suspended in incomplete Freund's adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis.

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in ...