We thank the Institut Pasteur Animalerie Centrale and the CB-UTechS facility members for their help. This work was supported financially by Institut Pasteur.

All the procedures agreed with the guidelines of the European Commission for the handling of laboratory animals, directive 86/609/EEC. They were approved by the ethical committees No. 59, by the CETEA/CEEA No. 089, under the number dap210067 and APAFIS #32382-2021070917055505 v1.
1. Preparation of the materials
<ol>
	<li>Store all antibodies (Table of Materials) at 4 &#176;C, protected from light exposure.</li>
	<li>DAPI stock solution (1 mg/mL): Resuspend t...

<ol>
	<li>Morais, L. H., Schreiber, H. L., Mazmanian, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gut+microbiota-brain+axis+in+behaviour+and+brain+disorders.">The gut microbiota-brain axis in behaviour and brain disorders.</a> Nature Reviews Microbiology. 19 (4), 241-255 (2021).</li><li>Deczkowska, A., Schwartz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+neuro-immune+communication+in+neurodegeneration:+Challenges+and+opportunities.">Targeting neuro-immune communication in neurodegeneration: Challenges and opportunities.</a> Journal of Experimental Medicine. 215 (11), 2702-2704 (2018).</li><li>Croese, T., Castellani, G., Schwartz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+compartmentalization+for+brain+surveillance+and+protection.">Immune cell compartmentalization for brain surveillance and protection.</a> Nature Immunology. 22 (9), 1083-1092 (2021).</li><li>Erny, D., 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=Host+microbiota+constantly+control+maturation+and+function+of+microglia+in+the+CNS.">Host microbiota constantly control maturation and function of microglia in the CNS.</a> Nature Neuroscience. 18 (7), 965-977 (2015).</li><li>Mrdjen, D., 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=High-dimensional+single-cell+mapping+of+central+nervous+system+immune+cells+reveals+distinct+myeloid+subsets+in+health,+aging,+and+disease.">High-dimensional single-cell mapping of central nervous system immune cells reveals distinct myeloid subsets in health, aging, and disease.</a> Immunity. 48 (2), 380-395 (2018).</li><li>Korin, B., 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=single-cell+characterization+of+the+brain's+immune+compartment.">single-cell characterization of the brain's immune compartment.</a> Nature Neuroscience. 20 (9), 1300-1309 (2017).</li><li>van Hove, H., 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=A+single-cell+atlas+of+mouse+brain+macrophages+reveals+unique+transcriptional+identities+shaped+by+ontogeny+and+tissue+environment.">A single-cell atlas of mouse brain macrophages reveals unique transcriptional identities shaped by ontogeny and tissue environment.</a> Nature Neuroscience. 22 (6), 1021-1035 (2019).</li><li>Ajami, B., 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=Single-cell+mass+cytometry+reveals+distinct+populations+of+brain+myeloid+cells+in+mouse+neuroinflammation+and+neurodegeneration+models.">Single-cell mass cytometry reveals distinct populations of brain myeloid cells in mouse neuroinflammation and neurodegeneration models.</a> Nature Neuroscience. 21 (4), 541-551 (2018).</li><li>Wolburg, H., Paulus, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choroid+plexus:+Biology+and+pathology.">Choroid plexus: Biology and pathology.</a> Acta Neuropathologica. 119 (1), 75-88 (2010).</li><li>Dani, N., 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=A+cellular+and+spatial+map+of+the+choroid+plexus+across+brain+ventricles+and+ages.">A cellular and spatial map of the choroid plexus across brain ventricles and ages.</a> Cell. 184 (11), 3056-3074 (2021).</li><li>Falc&#227;o, A. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+(Kolmer's)+epiplexus+cells.">The origin of (Kolmer's) epiplexus cells.</a> Histochemistry. 44 (1), 103-104 (1975).</li><li>Baruch, K., 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=CNS-specific+immunity+at+the+choroid+plexus+shifts+toward+destructive+Th2+inflammation+in+brain+aging.">CNS-specific immunity at the choroid plexus shifts toward destructive Th2 inflammation in brain aging.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (6), 2264-2269 (2013).</li><li>Kunis, G., 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=IFN-&#947;-dependent+activation+of+the+brain's+choroid+plexus+for+CNS+immune+surveillance+and+repair.">IFN-&#947;-dependent activation of the brain's choroid plexus for CNS immune surveillance and repair.</a> Brain. 136 (11), 3427-3440 (2013).</li><li>Prinz, M., Priller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+and+brain+macrophages+in+the+molecular+age:+From+origin+to+neuropsychiatric+disease.">Microglia and brain macrophages in the molecular age: From origin to neuropsychiatric disease.</a> Nature Reviews Neuroscience. 15 (5), 300-312 (2014).</li><li>Goldmann, T., 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=fate+and+dynamics+of+macrophages+at+central+nervous+system+interfaces.">fate and dynamics of macrophages at central nervous system interfaces.</a> Nature Immunology. 17 (7), 797-805 (2016).</li><li>Fung, I. 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Studies aiming to understand the immunological contributions to brain homeostasis and disease have mainly focused on cells residing within the brain parenchyma, neglecting brain borders such as CP, which are nevertheless crucial contributors to brain function2,3. The analysis of immune cell populations at CP is challenging due to the small size of CP, low numbers of resident immune cells, and complicated access to this tissue. Flow cytometry performed on total br...

Since the discovery of the blood-brain barrier by Paul Erhlich in the late 19th century, the brain has been considered virtually separated from the other organs and the bloodstream. Yet, this last decade has seen the emergence of the concept that brain function is shaped by various biological factors, such as gut microbiota and systemic immune cells and signals1,2,3,4. In parallel, other brain borders such as meninges and choroid plexuses (CP) have been identified as interfaces of active immune-brain cross talk rather than inert barrier tissues5,6,7,8.
The CP&#160;constitute the blood-cerebrospinal fluid barrier, one of the borders separating the brain and the periphery. They are located in each of the four ventricles of the brain, i.e., the third, the fourth, and both lateral ventricles, and are adjacent to areas involved in neurogenesis such as the subventricular zone and subgranular zone of the hippocampus3. Structurally, the CP are composed of a network of fenestrated blood capillaries enclosed by a monolayer of epithelial cells, which are interconnected by tight and adherens junctions9,10. Major physiological roles of the CP epithelium involve the production of cerebrospinal fluid, which flushes the brain from waste metabolites and protein aggregates, and the production and controlled blood-to-brain passage of various signaling molecules including hormones and neurotrophic factors11,12,13. Secreted molecules from CP shape brain&#39;s activity, i.e., by modulating neurogenesis and microglial function14,15,16,17,18,19, which makes CP crucial for brain homeostasis. CP also engage in various immune activities; whereas the main immune cell type in the brain parenchyma under non-pathological conditions is microglia, the diversity of CP immune cell populations is as broad as in peripheral organs3,7, suggesting that various channels of immune regulation and signaling are at work at the CP.
The space between the endothelial and epithelial cells, the CP stroma, is mainly populated by border-associated macrophages (BAM), which express pro-inflammatory cytokines and molecules related to antigen presentation in response to inflammatory signals3. Another subtype of macrophages, Kolmer&#39;s epiplexus cells, are present on the apical surface of the CP epithelium20. CP stroma is also a niche for dendritic cells, B cells, mast cells, basophils, neutrophils, innate lymphoid cells, and T cells which are mostly effector memory T cells able to recognize central nervous system antigens7,21,22,23,24. In addition, the composition and activity of immune cell populations at the CP changes upon systemic or brain perturbation, for example, during aging10,14,15,21,25, microbiota perturbation7, stress26, and disease27,28. Notably, these changes were suggested to indirectly shape brain function, i.e., a shift of CP CD4+ T cells towards Th2 inflammation occurs in brain aging and triggers immune signaling from the CP that may shape aging-associated cognitive decline14,15,21,25,29. Illuminating the properties of the CP immune cells would thus be crucial to better understand their regulatory function on CP epithelium physiology and secretion and thereby decipher their indirect impact on brain function under healthy and disease conditions.
CP are small structures that contain only a few immune cells. Their isolation requires microdissection after a preliminary step of perfusion; immune cells in the bloodstream would otherwise constitute major contaminants. This protocol aims to characterize the myeloid and T cell subsets of the CP using flow cytometry. This method identifies about 90% of the immune cell populations that compose mouse CP under non-inflammatory conditions, in accordance with recently published works using other methods to dissect immune CP heterogeneity7,10,28. This protocol could be applied to characterize changes in the CP immune cell compartment with disease and other experimental paradigms in vivo.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-mouse CD16/CD32</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>Albumin, bovine</td>
			<td>MP Biomedicals</td>
			<td>160069</td>
			<td>Blocking reagent</td>
		</tr>
		<tr>
			<td>APC anti-mouse CX3CR1</td>
			<td>BioLegend</td>
			<td>149008</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>APC anti-mouse TCRb</td>
			<td>BioLegend</td>
			<td>109212</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>APC-Cy7 anti-mouse CD4</td>
			<td>BioLegend</td>
			<td>100414</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>APC-Cy7 anti-mouse IA-IE</td>
			<td>BioLegend</td>
			<td>107628</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>BD FACSymphony A5 Cell Analyzer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometry analyzer</td>
		</tr>
		<tr>
			<td>BV711 anti-mouse Ly6C</td>
			<td>BioLegend</td>
			<td>128037</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Gibco</td>
			<td>17104-019</td>
			<td>Enzyme to dissociate CP tissue</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Scientific</td>
			<td>62248</td>
			<td>Live/dead marker</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td></td>
			<td></td>
			<td>Ion chelator</td>
		</tr>
		<tr>
			<td>fine scissors</td>
			<td>FST</td>
			<td>14058-11</td>
			<td>Dissection tool</td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD45</td>
			<td>BioLegend</td>
			<td>103108</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>Flow controller infusion inset</td>
			<td>CareFusion</td>
			<td>RG-3-C</td>
			<td>Blood perfusion inset</td>
		</tr>
		<tr>
			<td>FlowJo software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Analysis software</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>FST</td>
			<td>11018-12</td>
			<td>Dissection tool</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3149-10KU</td>
			<td>Anticoagulant</td>
		</tr>
		<tr>
			<td>Imalgene</td>
			<td>Boehringer Ingelheim</td>
			<td></td>
			<td>Ketamine, anesthesic</td>
		</tr>
		<tr>
			<td>OneComp eBeads</td>
			<td>Invitrogen</td>
			<td>01-1111-42</td>
			<td>Control beads to realize compensation</td>
		</tr>
		<tr>
			<td>PBS-/-</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>Buffer</td>
		</tr>
		<tr>
			<td>PBS+/+</td>
			<td>Gibco</td>
			<td>14040-091</td>
			<td>Buffer</td>
		</tr>
		<tr>
			<td>PE anti-mouse CD8a</td>
			<td>BioLegend</td>
			<td>100708</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>PE anti-mouse F4/80</td>
			<td>BioLegend</td>
			<td>123110</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>PE-Dazzle 594 anti-mouse CD11b</td>
			<td>BioLegend</td>
			<td>101256</td>
			<td>Flow cytometry antibody</td>
		</tr>
		<tr>
			<td>Rompun</td>
			<td>Bayer</td>
			<td></td>
			<td>Xylazine, anesthesic</td>
		</tr>
		<tr>
			<td>thin forceps</td>
			<td>Dumoxel Biology</td>
			<td>11242-40</td>
			<td>Dissection tool</td>
		</tr>
		<tr>
			<td>Vetergesic</td>
			<td>Ceva</td>
			<td></td>
			<td>Buprenorphin, analgesic</td>
		</tr>
	</tbody>
</table>

isolation and characterization of the immune cells from micro-dissected mouse choroid plexuses

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can indirectly shape brain function. At the interface between the brain and the systemic circulation, the choroid plexuses (CP), which constitute the blood-cerebrospinal fluid barrier, have been highlighted as a key site of periphery-to-brain communication. CP produce the cerebrospinal fluid, neurotrophic factors, and signaling molecules that can shape brain homeostasis. CP are also an active immunological niche. In contrast to the brain parenchyma, which is populated mainly by microglia under physiological conditions, the heterogeneity of CP immune cells recapitulates the diversity found in other peripheral organs. The CP immune cell diversity and activity change with aging, stress, and disease and modulate the activity of the CP epithelium, thereby indirectly shaping brain function. The goal of this protocol is to isolate murine CP and identify about 90% of the main immune subsets that populate them. This method is a tool to characterize CP immune cells and understand their function in orchestrating periphery-to-brain communication. The proposed protocol may help decipher how CP immune cells indirectly modulate brain function in health and across various disease conditions.

The flow cytometry analyses presented here successfully revealed the major subsets of myeloid and T cells (Figure 1 and Figure 2, respectively), and their relative total number per mouse in a highly reproducible manner (Figure 3).
The flow cytometry analysis of myeloid cells showed that CP are populated by CD11b+ CX3CR1+ F4/80high BAM, representing almost 80% of...

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Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

This study uses flow cytometry and two different gating strategies on isolated perfused mice brain choroid plexuses; this protocol identifies the main immune cell subsets that populate this brain structure.

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can ...

This protocol may be useful for addressing questions about the role of the choroid plexuses and peripheral immune cells in regulation of the brain in various contexts in mouse models. This protocol allows for in-depth quantitative and qualitative analysis of the unique immune cell populations in the choroid plexuses of mice. Dissection of the choroid plexuses can be difficult at the beginning due to their small size and transparent color.We recommend practicing on non-perfused animals first. To begin, position the brain dissected from a C57 black six mouse dorsal side up in a Petri dish and place the dish below the objectives of the binocular loops. While maintaining the brain in place using forceps, insert the two ends of another pair of forceps down through the midline between the hemispheres.Using the forceps, pull the cortex with the callosum and hippocampus away from the septum, exposing the lateral ventricle and a part of the third ventricle. Identify the lateral choroid plexus as a long veil lining the lateral ventricle and flaring at both ends. Then using two ends of thin forceps, collect the lateral choroid plexus.Be careful to collect the triangular posterior part hidden by the posterior fold of the hippocampus. Next, pull the cortex with the corpus callosum and hippocampus of the contralateral hemisphere away from the septum to expose the entire third ventricle and the opposite lateral ventricle. Using fine forceps, collect the third choroid plexus identified as a short structure with a granular surface aspect.Then collect the other lateral choroid plexus. Next, insert the two ends of the forceps down between the cerebellum and midbrain and detach the cerebellum from the pons and medulla to expose the fourth ventricle. Then identify the fourth choroid plexus as a long globular structure with a granular surface aspect that lines the fourth ventricle from the lateral right end to the left end between the cerebellum and medulla.Using fine forceps, collect the fourth choroid plexus. After incubating all collected choroid plexuses with collagenase IV, gently pipette up and down around 10 times to finalize the dissociation. Then fill the tube to 1.5 milliliters with MACS buffer to stop the collagenase IV activity.Next, centrifuge the cells and discard the supernatant before washing the cells with 1.5 milliliters of MACS buffer. After adding the appropriate compensation beads and antibody solutions, incubate the samples on ice for 30 to 45 minutes, protecting them from light exposure. Then fill the tubes with 1.5 milliliters of MACS buffer and centrifuge the cells and compensation beads.After resuspending the cells and compensation beads in 500 microliters of MACS buffer, filter the cells through a 70 micron strainer. After turning on the flow cytometer and the software, select forward scatter area versus side scatter area and gate for cells based on size. Then create a daughter gate for single cells with forward scatter area versus forward scatter height.Next, create a daughter gate for live cells with DAPI versus forward scatter area to exclude the DAPI positive cells. Then create a daughter gate for CD45 positive immune cells with CD45 versus forward scatter area. Now, create a daughter gate from the CD45 positive cells and select F480 versus CD11b to identify the CD11b positive F480 positive and the CD11b positive F480 negative populations.Then create a daughter gate for CD11b positive F480 positive cells and select CX3CR1 versus IA-IE to identify both CX3CR1 positive IA-IE positive border-associated macrophages and CX3CR1 positive IA-IE negative macrophages. Next, create a daughter gate for CD11b positive F480 positive CX3CR1 positive IA-IE negative macrophages and select F480 versus CD11b to identify both the CD11b high F480 intermediate CX3CR1 positive IA-IE negative Kolmer's epiplexus macrophages and the CD11b positive F480 high CX3CR1 positive IA-IE negative border-associated macrophages. Then from the CD11b positive F480 negative population, gate for Ly6C versus IA-IE.Select both IA-IE positive Ly6C negative population and IA-IE negative Ly6C positive cells that mainly correspond to monocytes or neutrophils. Finally, create a daughter gate for CD11b positive F480 negative IA-IE positive Ly6C negative population and select CD11b versus CX3CR1. Then identify both CD11b high CX3CR1 low and CD11b positive CX3CR1 negative populations.For T-cell gating, after excluding the DAPI positive cells as described earlier, create a daughter gate for CD45 positive immune cells with CD45 versus FSC-A, then create a daughter gate from the CD45 positive cells and select TCR beta versus CD11b. Exclude the CD11b positive myeloid cells and select the TCR positive CD11b negative T-cell population. Finally, create a daughter gate for the TCR beta positive CD11b negative population and select CD4 versus CD8a.Identify both CD8 negative and CD4 positive T-cells and CD8 positive CD4 negative T-cells. The flow cytometry analyses demonstrated here successfully revealed major subsets of myeloid and T-cells and their relative total number per mouse in a highly reproducible manner. Flow cytometry analysis of the myeloid cells show that the choroid plexus is populated by CD11b positive CX3CR1 positive F480 high border-associated macrophages, representing 80%of the CD45 positive immune cells at the choroid plexus.The CD11b high F480 intermediate CX3CR1 positive IA-IE negative population of Kolmer's epiplexus macrophages was also identified. Among the CD11b positive F480 negative immune cells, two different populations of Ly6C negative IA-IE positive cells were characterized and likely correspond to dendritic cells. The analysis and gating strategy of T-cells highlighted the presence of the minor population of CD45 positive CD11b negative TCR beta positive cells.Among them, about 30-50 CD8 negative CD4 positive and CD8 positive CD4 negative T-cells per mouse populated the choroid plexus under physiological conditions. It is important to control for the quality of the perfusion since any blood contamination may blur the immune composition of the choroid plexus. This method can be followed by sorting of selected cell populations for further analysis such as bulk or single-cell RNA sequencing.

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