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 <e...

<ol>
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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. 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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. 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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 ...

The protocol we present here will be helpful for us to understand how lymphocytes are involved in the development of the central nervous system autoimmune disease. With this method, lymphocytes in the brain can be studied studied on a cell by cell basis. This protocol can also be applied to other models and central nervous system disease that needed to analyze the characteristics and functions of immune cells.In this video, the protocol can easily demonstrate how to induce model and isolate single cells in the brain. After confirming a lack of response to pedal reflex, use a one milliliter syringe to subcutaneously inject anesthetized C57BL/6 mice with 100 micrograms of emulsified MOG 35-55 in 100 microliters of complete Freund's adjuvant into two different sites of the back near the neck. On the same day and on day two postimmunization, intraperitoneally inject the mice with 200 microliters of one nanogram per microliter pertussis toxin working solution.After the second injection, transfer the mice back to their home cage with a warming pad and monitoring until full recumbency. The next day and throughout the course of the disease, examine and grade all of the mice in a blinded manner for the neurological signs outlined in the table and for any changes in weight. At the appropriate experimental endpoint, cut the cranium carefully from the nose to the neck and transfer the brain of each animal from the cranial box into individual 50 milliliter tubes containing 10 milliliters of RPMI medium.Mix the tubes well to remove the adherent red blood cells and remove the medium by aspiration. Add 10 milliliters of fresh medium to each tube and transfer the brains into individual 100 millimeter dishes. Finally chop each brain with a razor and use a plastic pipette to transfer as much tissue from one dish at a time and six milliliters of medium into an icecold seven milliliter centered glass homogenizer.Grind the brain using the loose plunger of the pestle before using the tight plunger to bring the tissue until the suspension is homogeneous. Then, pour the resulting tissue slurry into a pre-chilled 15 milliliter conical tube on ice. When all the samples have been homogenized, adjust the volume in each tube to seven milliliters with fresh medium, before adding each tissue suspension to a new chilled 15 milliliter tube containing three milliliters of 100%basement membrane matrix per tube.Mix by inversion a few times and use a three milliliter pipette to carefully and slowly add one milliliter of 70 percent density gradient solution under each tissue solution sample. Separate the cells by density gradient centrifugation and remove almost all of the top layer, taking care to completely remove all of the myelin. Transfer the interface into a new 15 milliliter tube and adjust the volume to 10 milliliters with fresh medium.Then centrifuge the samples again and remove the supernatant before resuspending the pellets in about 100 microliters of medium per tube. For flow cytometric analysis of the harvested brain cells, allocate approximately two times 10 to the six cells in 100 microliters of medium into individual wells of a 96-well plate. When all of the cells have been plated, add 100 microliters of 500X cell stimulation cocktail plus protein transport inhibitors to the wells and place the plate in the cell culture incubator for four hours.At the end of the incubation pool the cells at the bottom of the wells by centrifugation and resuspend the pellets in 100 microliters of flow cytometry buffer per well. Pre-incubate the cells with three microliters of Anti-Mouse CD16/CD32 Fc Block for 10 minutes at four degrees Celsius before staining. To block any nonspecific Fc-mediated interactions.At the end of the incubation, add the antibody cocktail of interest to each well and place the plate at four degrees Celsius protected from light. After 30 minutes, wash the wells with 100 microliters of flow cytometry buffer per well and sediment the cells by centrifugation. After discarding the supernatants, add 200 microliters of intracellular fixation buffer to each well.After a 30 to 60 minute incubation at room temperature, collect the samples by centrifugation and resuspend the pellets in 200 microliters of 1X permeability buffer per well. Centrifuge the samples again, and after discarding the supernatant, resuspend the pellets in 100 microliters with fresh permeabilization buffer. Next, add the appropriate antibodies for the detection of any intracellular antigens of interest for a 30 minute incubation at four degrees Celsius, protected from light.At the end of the incubation, add 100 microliters of 1X permeabilization buffer to each well and spin down the samples by centrifugation. Then, resuspend the stain cells in 200 microliters of flow cytometry staining buffer and analyze the cells by flow cytometry according to standard protocols. A typical clinical course of EAE should result in a disease curve and change in body weight as illustrated.C57BL/6 mice immunized with MOG 35-55 usually start to develop disease symptoms around day 10 to 12 and achieve the peak of disease around day 15 to 21. Before the onset of disease, the body weight of immunized mice gradually increases before decreasing in correlation with the increasing disease symptoms The mice demonstrate the lowest body weight at the peak of EAE, with body weights recovering slightly as the clinical symptoms decrease. The mice usually do not fully recover however and typically go on to develop a monophasic chronic disease pathology.As this representative flow analysis illustrates, interferon gamma-producing Th1 and IL17-producing Th17 cells are significantly increased in EAE mice. The most important step is 3.10. The operator should transfer middle layer and the light, being carefully, take as few other layers as possible.Following these protocol, we can further T lymphocytes for further transcription analysis to study

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Immunology and Infection

6.8K visualizações.  Shanghai Jiao Tong University School of Medicine. O protocolo que apresentamos aqui será útil para entendermos como os linfócitos estão envolvidos no desenvolvimento da doença autoimune do sistema nervoso central. Com este método, linfócitos no cérebro podem ser estudados em uma célula por célula. Este protocolo também pode ser aplicado a outros modelos e doenças do sistema nervoso central que precisavam analisar as características e funções das células imunes.Neste vídeo, o protocolo pode facilmente demonstrar como i...