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 the powder in PBS-/- (Table of Materials), aliquot, and store at -20 &#176;C.</li>
	<li>DAPI working solution (0.1 mg/mL): Dilute DAPI stock solution with PBS-/- at a ratio of 1:10.</li>
	<li>Magnetic-activated cell sorting (MACS) buffer: Prepare 2 mM ethylene diamine tetraacetic acid (EDTA) and 0.5% bovine serum albumin (BSA) (Table of Materials) in PBS-/-.</li>
	<li>Collagenase IV stock solution (20 U/&#956;L): Resuspend the powder in PBS+/+ (Table of Materials), aliquot, and store at -20 &#176;C. 
	NOTE: Collagenase IV requires MgCl2 to be fully active; do not freeze-thaw Collagenase IV solution.</li>
	<li>Anesthetic-analgesic solution: Mix 150 &#956;L of ketamine (150 mg/kg), 25 &#956;l of xylazine (5 mg/kg), and 330 &#956;l of buprecare (0.1 mg/kg) in 1 mL of PBS-/-. 
	NOTE: Prepare anesthetic-analgesic solution extemporaneously and do not keep it longer than a day.</li>
	<li>Heparin solution (100 U/mL): Resuspend the powder in PBS-/-.</li>
	<li>Prepare the infusion inset system connecting a tube to a decanter Erlenmeyer filled with PBS-/-. Connect a 23&#160;G needle to the extremity of the infusion tube. Open the infusion inset and let the PBS run until there are no bubbles in the tube.</li>
	<li>Prepare the binocular loupe equipped with a light.</li>
</ol>
2. Housing of C57BL/6 mice
<ol>
	<li>For the analysis of CP myeloid or T cells, use four mice for each and pool the four CP (two from each lateral ventricle, the third ventricle and the fourth&#160;ventricle CP; for eight mice in total). Not pooling CP carries the risk of failing to detect the rare immune populations in CP due to their low abundance.</li>
	<li>Let the mice acclimatize for at least 7 days prior to any experimentation. Keep the mice under pathogen-free conditions at constant temperature and humidity, in a 12/12 h or 14/10 h light/dark cycle, with water and standard pellet food ad libitum.</li>
</ol>
3.&#160;PBS perfusion and brain dissection
<ol>
	<li>Weight each mouse and inject 10 &#956;L/g of the anesthetic-analgesic mix solution (step 1.6) intraperitoneally.</li>
	<li>Wait around 30 min for efficient analgesia (check for the depth of anesthesia by pinching the mouse&#39;s digits).</li>
	<li>Position the anesthetized mouse flat on its back, on the dissection support, and tape its palms to the dissection support.</li>
	<li>Pinching the skin of the animal with forceps and using scissors, open the abdomen, the diaphragm, and the thorax to expose the heart. 
	NOTE: When the thorax is opened, the brain becomes anoxic. One must proceed precisely&#160;and swiftly through the next steps of the perfusion.</li>
	<li>Inject 20 &#956;L of 100 U/mL heparin solution directly into the left ventricle.</li>
	<li>With fine scissors, make an incision of at least 3 mm in the right atrium to allow the blood to flow out of the body.</li>
	<li>Immediately after, insert the 23 G needle placed at the extremity of the infusion tube through the tip of the left ventricle.</li>
	<li>Open the infusion system at the maximum and wait for complete perfusion: Using a 23 G needle, at a flow rate of about 6 mL/min, the perfusion is complete in 3 min. 
	NOTE: The even discoloration of organs such as the liver attests for perfusion efficacy.</li>
	<li>Close the infusion system and remove the needle from the heart ventricle.</li>
	<li>Remove the tape from the mouse&#39;s palms. Place the mouse in a ventral position.</li>
	<li>Pinching the skin of the animal with forceps and using scissors, remove the skin of the top of the head from the eyes to the ears.</li>
	<li>With the help of scissors, cut the skull first between the eyes and then laterally from each eye to the spinal cord just above the masseter muscles. For this, use the extremity of scissors and proceed gently to avoid damaging the brain with the scissors.</li>
	<li>Open the skull with forceps by pinching it from the extremity between the eyes.</li>
	<li>Use scissors to cut the spinal cord and extract the brain with forceps, placing their two points on the lateral side of the brain to tilt it and place it in a Petri dish filled with ice-cold PBS+/+. The CP are then immediately collected. 
	NOTE: Check for discoloration of the brain to verify perfusion efficacy.</li>
</ol>
4. Dissection of the Choroid Plexus from the brain
<ol>
	<li>Position the brain dorsal side up in the Petri dish and below the objectives of the binocular loupes.</li>
	<li>With the help of forceps to maintain the brain in place, insert the two ends of another forceps down through the midline between the hemispheres.</li>
	<li>Use the forceps to pull the cortex with the callosum and hippocampus away from the septum, exposing the lateral ventricle and a part of the third ventricle.</li>
	<li>Identify the lateral CP as a long veil lining the lateral ventricle that is flaring at both ends. Use the two ends of a thin forceps to catch the lateral CP. Be careful to collect the triangular posterior part that may be hidden by the posterior fold of the hippocampus.</li>
	<li>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.</li>
	<li>Collect with fine forceps the third CP, which can be identified as a short structure with a granular surface aspect.</li>
	<li>Collect the other lateral CP.</li>
	<li>Insert the two ends of a forceps down between the cerebellum and midbrain. Detach the cerebellum from the pons and medulla to expose the fourth ventricle.</li>
	<li>Identify the fourth CP 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 the medulla. Collect the fourth CP with fine forceps. 
	NOTE: Silane-base coating of forceps may prevent stickiness of CP on forceps.</li>
	<li>Repeat steps 3.1-4.9 for each of the other seven mice. In the meantime, the collected CP can be kept temporally in a tube placed on ice.</li>
</ol>
5. Preparation of samples for flow cytometry analysis
<ol>
	<li>Fill the tube containing all dissected CP to 750 &#956;L of PBS+/+. 
	NOTE: The deposition of dissected CP with thin forceps in the tube brings a small volume of PBS+/+. Rather complete the existing volume to 750 &#956;L than remove and replace it with fresh 750 &#956;L of PBS+/+ to avoid a possible loss of the CP tissue.</li>
	<li>Add 15 &#956;L of 20 U/&#956;L Collagenase IV stock solution (see step 1.5) for a 400 U/mL final concentration. 
	NOTE: DNAse I (150 &#956;g/mL) may prevent excessive cell clumping.</li>
	<li>Incubate CP with Collagenase IV at 37 &#176;C under mild agitation (300 RPM) for 45 min.</li>
	<li>Gently pipette up and down around 10 times to finalize the CP dissociation.</li>
	<li>Fill CP tube to 1.5 mL with MACS buffer (see recipe step 1.4) to stop collagenase IV activity. 
	NOTE: Collagenase IV activity is stopped at low temperature and inhibited by serum albumin.</li>
	<li>Centrifuge the cells at 500 x g, for 5 min, at 4 &#176;C. Discard the supernatant and wash the cells with 1.5 mL of MACS buffer.</li>
	<li>Centrifuge the cells at 500 x g for 5 min, at 4 &#176;C. Discard the supernatant and resuspend cells in 220 &#956;L of MACS buffer.</li>
	<li>Separate an aliquot of 10 &#956;L from the cell suspension to be used as unstained control (next step 5.18).</li>
	<li>Separate an aliquot of 10 &#956;L from the cell suspension to be used as DAPI-mono-stained control (next step 5.12).</li>
	<li>Preincubate the remaining 200 &#956;L with anti-mouse CD16/CD32 blocking antibody (1:100) for 20 min, at 4 &#176;C, to block nonspecific Fc-mediated interactions.</li>
	<li>Split the cells into two 100 &#956;L tubes (one for myeloid cells analysis, the other for T cells analysis) (next step 5.14).</li>
	<li>Take the sample with 10 &#956;L of cells for the DAPI-mono-stained control (step 5.9) and fill it up to 100 &#956;L with MACS buffer (next step 5.14).</li>
	<li>Prepare eleven new tubes containing 100 &#956;L of MACS for the antibody mono-stained (step 5.14.4) and all-stained controls (step 5.14.5) and add a drop of compensation beads into each. 
	NOTE: The compensation controls will allow setting up the voltage of fluorescent detection laser and assess the potential unspecific signal detection of fluorescent dye in the other channels that will be further compensated.</li>
	<li>Antibody incubation of samples:
	<ol>
		<li>Myeloid sample: Add 0.1 mg/mL DAPI (1:100) (see step 1.3), FITC anti-mouse CD45 (1:100), PE-Dazzle 594 anti-mouse CD11b (1:100), APC anti-mouse CX3CR1 (1:100), BV711 anti-mouse Ly6C (1:100), PE anti-mouse F4/80 (1:100), and APC-Cy7 anti-mouse IA-IE (1:100).</li>
		<li>T cells sample: Add 0.1 mg/mL DAPI (1:100), FITC anti-mouse CD45 (1:100), PE-Dazzle 594 anti-mouse CD11b (1:100), APC anti-mouse TCR&#946; (1:100), PE anti-mouse CD8a (1:100), and APC-Cy7 anti-mouse CD4 (1:50).</li>
		<li>DAPI-mono-stained control (from step 5.12): Add 0.1 mg/mL DAPI (1:100).</li>
		<li>Antibody mono-stained compensation beads: Add each of the nine previously used antibodies for myeloid and T cells samples (same dilution), separately (one for each tube) to the nine tubes containing the beads. 
		NOTE: The mono-stained compensation controls will allow the determination of positive peaks of fluorescence for each of the used fluorescent markers during the compensation step. The analyzer will compare them to the negative signal observed in the unstained control CP cells.</li>
		<li>Antibody all-stained compensation beads: Add all the antibodies used for myeloid staining to one tube and those used for T cells staining to another. 
		NOTE: The all-stained compensation controls will allow setting up the voltage of each fluorescent laser detection and assess the potential unspecific signal detection of fluorescent dye in the other channels.</li>
		<li>Incubate for 30-45 min on ice, protected from light exposure.</li>
	</ol>
	</li>
	<li>Fill all tubes up to 1.5 mL with MACS buffer. Centrifuge the cells and compensation beads at 500 x g, for 5 min, at 4 &#176;C.</li>
	<li>Discard the supernatant and wash the cells and compensation beads in 1.5 mL of MACS buffer.</li>
	<li>Centrifuge the cells and compensation beads at 500 x g, for 5 min, at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend the cells and compensation beads in 500 &#956;L of MACS buffer. Fill unstained control (step 5.8) up to 500 &#956;L with MACS buffer.</li>
	<li>Filter each tube with CP cells through a 70 &#956;m strainer (unstained CP, DAPI mono-stained control, and myeloid and T cells samples).</li>
</ol>
6. Flow cytometry
NOTE: The flow cytometer used in this protocol is equipped with the following 5 lasers: a 355 nm UV laser, a 405 nm Violet laser, a 488 nm Blue laser, a 561 nm Yellow-Green laser and a 637 nm Red laser.
<ol>
	<li>Perform the daily quality control checks on the cytometer using the cytometer setup, tracking (CST) interface, and the CST control beads to ensure consistency between analyses, instrument quality, and consistent target fluorescence intensities.</li>
	<li>To set up the flow cytometry experiment, create a new experiment with bivariate plots and histograms. Ensure inclusion of a forward scatter area (FSC-A) and side scatter area (SSC-A) plots as well as histogram plots for each color to monitor the acquisition.</li>
	<li>Define the cell morphology by setting up the PMT voltage for FSC-A and SSC-A parameters on unstained control CP cells.</li>
	<li>Define the voltages of each fluorescent marker by setting up the negative peaks of the fluorochromes on unstained control CP cells and the positive peaks of the fluorochromes using compensation beads stained with all antibodies and ensuring that the detector signals are not off the scale and are not too strongly cross-detected in other channels.</li>
	<li>Create a compensation matrix to analyze each of the single-color compensation controls. After gating on appropriate FSC-A/SSC-A populations, check single-stained controls on histogram plots and record compensation controls. After recording all single-stained samples, calculate the compensation matrix.</li>
	<li>Record all events detected for both myeloid and T cell populations.</li>
</ol>
7. CP myeloid cells gating
<ol>
	<li>Select FSC-A vs. SSC-A and gate for cells (based on size).</li>
	<li>Create a daughter gate for single cells with FSC-A vs. forward scatter height (FSC-H).</li>
	<li>Create a daughter gate for live cells with DAPI vs. FSC-A to exclude DAPI+ cells.</li>
	<li>Create a daughter gate for CD45+ immune cells with CD45 vs. FSC-A.</li>
	<li>Create a daughter gate from the CD45+ cells and select CD11b vs. F4/80, identify the following two groups: the CD11b+ F4/80+ and the CD11b+ F4/80- populations (next step 7.8).</li>
	<li>Create a daughter gate for CD11b+ F4/80+ cells and select CX3CR1 vs. IA-IE. Identify both CX3CR1+ IA-IE+ BAM that are further denominated as CD11b+ F4/80high CX3CR1+ IA-IE+ BAM, and CX3CR1+ IA-IE- macrophages.</li>
	<li>Create a daughter gate for CD11b+ F4/80+ CX3CR1+ IA-IE- macrophages and select F4/80 vs. CD11b. Identify both the CD11bhigh F4/80intermediate and the CD11b+ F4/80high populations that are further called CD11bhigh F4/80intermediate CX3CR1+ IA-IE- Kolmer&#39;s epiplexus macrophages and CD11b+ F4/80high CX3CR1+ IA-IE- BAM, respectively. 
	NOTE: CD11bhigh F4/80intermediate CX3CR1+ IA-IE- and CD11b+ F4/80high CX3CR1+ IA-IE- cells appeared a bit intermixed between F4/80intermediate-F4/80high and CD11bhigh-CD11b+ levels.</li>
	<li>From the CD11b+ F4/80- population (step 7.5), gate for Ly6C vs. IA-IE. Select both IA-IE+ Ly6C- population and IA-IE- Ly6C+ cells that mainly correspond to monocytes/neutrophils. 
	NOTE: A high presence of IA-IE- Ly6C+ cells likely reflects problems in the perfusion efficacy of animals without an inflammatory condition; their abundance should be low.</li>
	<li>Create a daughter gate for CD11b+ F4/80- IA-IE+ Ly6C- population and select CD11b vs. CX3CR1, identify both CD11bhigh CX3CR1low and CD11b+ CX3CR1- populations.</li>
</ol>
8. CP T cells gating
<ol>
	<li>Select FSC-A vs. SSC-A and gate for cells (based on size).</li>
	<li>Create a daughter gate for single cells with FSC-A vs. forward scatter height (FSC-H).</li>
	<li>Create a daughter gate for live cells with DAPI vs. FSC-A to exclude DAPI+ cells.</li>
	<li>Create a daughter gate for CD45+ immune cells with CD45 vs. FSC-A.</li>
	<li>Create a daughter gate from the CD45+ cells and select TCR&#946; vs. CD11b. Exclude CD11b+ myeloid cells and select TCR&#946;+ CD11b- T cells population.</li>
	<li>Create a daughter gate for TCR&#946;+ CD11b- population and select CD4 vs. CD8a. Identify both CD8- CD4+ T cells and CD8+ CD4- T cells.</li>
</ol>

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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=Activation+of+group+2+innate+lymphoid+cells+alleviates+aging-associated+cognitive+decline.">Activation of group 2 innate lymphoid cells alleviates aging-associated cognitive decline.</a> Journal of Experimental Medicine. 217 (4), (2020).</li><li>Kertser, A., 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=Corticosteroid+signaling+at+the+brain-immune+interface+impedes+coping+with+severe+psychological+stress.">Corticosteroid signaling at the brain-immune interface impedes coping with severe psychological stress.</a> Science Advances. 5, 4111 (2019).</li><li>Shechter, R., 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=Recruitment+of+beneficial+M2+macrophages+to+injured+spinal+cord+is+orchestrated+by+remote+brain+choroid+plexus.">Recruitment of beneficial M2 macrophages to injured spinal cord is orchestrated by remote brain choroid plexus.</a> Immunity. 38 (3), 555-569 (2013).</li><li>Yang, A. C., 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=Dysregulation+of+brain+and+choroid+plexus+cell+types+in+severe+COVID-19.">Dysregulation of brain and choroid plexus cell types in severe COVID-19.</a> Nature. 595 (7868), 565-571 (2021).</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=PD-1+immune+checkpoint+blockade+reduces+pathology+and+improves+memory+in+mouse+models+of+Alzheimer's+disease.">PD-1 immune checkpoint blockade reduces pathology and improves memory in mouse models of Alzheimer's disease.</a> Nature Medicine. 22 (2), 135-137 (2016).</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=Breaking+immune+tolerance+by+targeting+Foxp3++regulatory+T+cells+mitigates+Alzheimer's+disease+pathology.">Breaking immune tolerance by targeting Foxp3+ regulatory T cells mitigates Alzheimer's disease pathology.</a> Nature Communications. 6, 7967 (2015).</li><li>Rodr&#237;guez-Rodr&#237;guez, N., Flores-Mendoza, G., Apostolidis, S. A., Rosetti, F., Tsokos, G. C., Crisp&#237;n, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCR-&#945;/&#946;+CD4+&#8722;+CD8+&#8722;+double+negative+T+cells+arise+from+CD8+++T+cells.">TCR-&#945;/&#946; CD4 &#8722; CD8 &#8722; double negative T cells arise from CD8 + T cells.</a> Journal of Leukocyte Biology. 108 (3), 851-857 (2020).</li><li>Schafflick, 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=Single-cell+profiling+of+CNS+border+compartment+leukocytes+reveals+that+B+cells+and+their+progenitors+reside+in+non-diseased+meninges.">Single-cell profiling of CNS border compartment leukocytes reveals that B cells and their progenitors reside in non-diseased meninges.</a> Nature Neuroscience. 24 (9), 1225-1234 (2021).</li><li>Quintana, E., 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=DNGR-1++dendritic+cells+are+located+in+meningeal+membrane+and+choroid+plexus+of+the+noninjured+brain.">DNGR-1+ dendritic cells are located in meningeal membrane and choroid plexus of the noninjured brain.</a> GLIA. 63 (12), 2231-2248 (2015).</li><li>Kabashima, 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=Biomarkers+for+evaluation+of+mast+cell+and+basophil+activation.">Biomarkers for evaluation of mast cell and basophil activation.</a> Immunological Reviews. 282 (1), 114-120 (2018).</li><li>Li, Q., Barres, B. A. <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+macrophages+in+brain+homeostasis+and+disease.">Microglia and macrophages in brain homeostasis and disease.</a> Nature Reviews Immunology. 18 (4), 225-242 (2018).</li><li>Borst, K., Dumas, A. A., Prinz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia:+Immune+and+non-immune+functions.">Microglia: Immune and non-immune functions.</a> Immunity. 54 (10), 2194-2208 (2021).</li></ol>

The authors declare no competing financial interests.

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><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...

Watch this Scientific Journal Video about Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses at JoVE.com

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 ...

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6.7K Views.  Institut Pasteur. 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.

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

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

Immunology and Infection

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Neuroscience

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during development, homeostasis, and disease. Although macrophages have been studied in detail for decades, specialized features of microglia, the tissue-resident macrophages of the CNS, have remained largely mysterious, in part due to limitations in the ability to recapitulate mature microglial properties in culture. Here, we illustrate a straightforward procedure for the rapid isolation of pure microglia from the mature rodent brain. We also describe serum-free culture conditions that support high levels of microglial viability over time. Microglia cultured under these defined-medium conditions exhibit elaborate ramified processes and dynamic surveillance behavior. We illustrate some effects of serum exposure on cultured microglia and discuss how these serum-free cultures compare to both serum-exposed cultures as well as microglia in vivo.

Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions.

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Microdissection and Whole Mount Scanning Electron Microscopy Visualization of Mouse Choroid Plexus

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in neuroscience. As it is becoming increasingly clear that this tiny structure plays a crucial role in health and disease of the central nervous system (CNS), it is of utmost importance to properly dissect the CP out of the brain ventricles in a way that allows downstream processing, ranging from functional to structural analysis. Here, isolation of the lateral and fourth brain ventricle mouse CP without the need for specialized tools or equipment is described. This isolation technique preserves the viability, function, and structure of cells within the CP. On account of its high vascularization, the CP can be visualized floating inside the ventricular cavities of the brain using a binocular microscope. However, transcardial perfusion required for downstream analysis can complicate the identification of the CP tissue. Depending on the further processing steps (e.g., RNA and protein analysis), this can be solved by visualizing the CP via transcardial perfusion with bromophenol blue. After isolation, the CP can be processed using several techniques, including RNA, protein, or single cell analysis, to gain further understanding on the function of this special brain structure. Here, scanning electron microscopy (SEM) on whole mount CP is used to get an overall view of the structure.

The choroid plexus (CP), an understudied tissue in neuroscience, plays a key role in health and disease of the central nervous system. This protocol describes a microdissection technique for isolating the CP and the use of scanning electron microscopy to obtain an overall view of its cellular structure.

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in ...

Obtaining Single-Cell Suspensions from Murine Brain and Skull Bone Marrow

Isolating Immune Cells from Mouse Brain and Skull

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

Author Spotlight: Investigating the Complex Immune Response Following Brain Ischemia

To investigate the immune response to brain disorders, one common approach is to analyze changes in immune cells. Here, two simple and effective protocols are provided for isolating immune cells from murine brain tissue and skull bone marrow.

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only ...

Preparing a Single-Cell Suspension of Immune Cells from Murine Brain Tissue

Source: DiSano, K. D., et al. <a href="https://app.jove.com/v/61166">Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune Cells.</a> J. Vis. Exp. (2020).
The video demonstrates the preparation of a single-cell suspension of immune cells from a murine brain tissue. It involves enzymatic digestion to release cells from the brain tissue, followed by density gradient centrifugation to isolate the immune cells based on their densities to obtain the single-cell suspension.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparing single-cell ...