Name: flow cytometric analysis of lymphocyte infiltration in central nervous system during experimental autoimmune encephalomyelitis
Uploaded: 2020-11-17T13:00:00
Description: Watch this Scientific Journal Video about Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis at JoVE.com

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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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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