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>

<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. M., Marques, F., Novais, A., Sousa, N., Palha, J. A., Sousa, 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=The+path+from+the+choroid+plexus+to+the+subventricular+zone:+Go+with+the+flow.">The path from the choroid plexus to the subventricular zone: Go with the flow.</a> Frontiers in Cellular Neuroscience. 6, (2012).</li><li>Shipley, F. 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=Tracking+calcium+dynamics+and+immune+surveillance+at+the+choroid+plexus+blood-cerebrospinal+fluid+interface.">Tracking calcium dynamics and immune surveillance at the choroid plexus blood-cerebrospinal fluid interface.</a> Neuron. 108 (4), 623-639 (2020).</li><li>Mazucanti, C. 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=Release+of+insulin+produced+by+the+choroids+plexis+is+regulated+by+serotonergic+signaling.">Release of insulin produced by the choroids plexis is regulated by serotonergic signaling.</a> JCI Insight. 4 (23), (2019).</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=Aging-induced+type+I+interferon+response+at+the+choroid+plexus+negatively+affects+brain+function.">Aging-induced type I interferon response at the choroid plexus negatively affects brain function.</a> Science. 346 (6205), 89-93 (2014).</li><li>Deczkowska, 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=Mef2C+restrains+microglial+inflammatory+response+and+is+lost+in+brain+ageing+in+an+IFN-I-dependent+manner.">Mef2C restrains microglial inflammatory response and is lost in brain ageing in an IFN-I-dependent manner.</a> Nature Communications. 8 (1), (2017).</li><li>Silva-Vargas, V., Maldonado-Soto, A. R., Mizrak, D., Codega, P., Doetsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+niche+signals+from+the+choroid+plexus+regulate+adult+neural+stem+cells.">Age-dependent niche signals from the choroid plexus regulate adult neural stem cells.</a> Cell Stem Cell. 19 (5), 643-652 (2016).</li><li>Iliff, J. J., 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=Impairment+of+glymphatic+pathway+function+promotes+tau+pathology+after+traumatic+brain+injury.">Impairment of glymphatic pathway function promotes tau pathology after traumatic brain injury.</a> Journal of Neuroscience. 34 (49), 16180-16193 (2014).</li><li>Redzic, Z. B., Preston, J. E., Duncan, J. A., Chodobski, A., Szmydynger-Chodobska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+choroid+plexus-cerebrospinal+fluid+system:+From+development+to+aging.">The choroid plexus-cerebrospinal fluid system: From development to aging.</a> Current Topics in Developmental Biology. 71, 1-52 (2005).</li><li>da Mesquita, 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=Functional+aspects+of+meningeal+lymphatics+in+ageing+and+Alzheimer's+disease.">Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease.</a> Nature. 560 (7717), 185-191 (2018).</li><li>Schwarze, E. -. 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. T. 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 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...

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 ...

isolation-characterization-immune-cells-from-micro-dissected-mouse

Research

JoVE Journal

Biology

6.6K 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 Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of &#62;95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...