Name: Isolation of Pericytes from Mouse Cortical Tissue Using FACS for Single-cell Sequencing (Video) | JoVE Journal
Uploaded: 2026-01-30T14:30:00.000+00:00
Duration: 10 min 50 s
Description: Learn to isolate mouse brain pericytes using FACS with CD13+/CD31- sorting for single-cell RNA sequencing. This optimized protocol enables high-quality analysis of pericyte heterogeneity in neurovascular disease research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by the Beijing Natural Science Foundation (Grant No. 7242222), the Provincial Science and Technology Plan Project of Hebei Province (Grant No. 203777110D), and the Microcirculation Research Institute of Peking Union Medical College, Chinese&#160;Academy of Medical Sciences (Grant No. CAMS-IM-2023-02).

In this study, we used three adult C57BL/6J mice (3 months old, 20-25 g). All mice were housed under a 12 h light/dark cycle with strictly controlled temperature and humidity, with free access to food and water prior to experiments. All experimental procedures involving laboratory animals were approved by the Laboratory Animal Care and Ethics Committee of the Institute of Microcirculation, Chinese Academy of Medical Sciences, and Peking Union Medical College, and were conducted in strict accordance with relevant animal welfare guidelines.
1. Reagent preparation
<ol>
	<li>30% (wt/vol) Glucose Solution: Dissolve 15 g glucose in 50 mL of UltraPure DNase/RNase-free dH2O. To ensure complete dissolution, first add glucose to 20 mL of UltraPure DNase/RNase-free dH2O and mix thoroughly. Then add UltraPure DNase/RNase-free dH2O to a final volume of 50 mL. Filter the solution and store at 4 &#176;C for up to 6 months.</li>
	<li>DNase I (10 mg/mL ): Dissolve 50 mg DNase I in 5 mL of sterile ddH2O. The stock solution can be stored at -20 &#176;C for up to 2 months. 
	NOTE: Repeated freeze-thaw cycles should be strictly avoided to prevent enzymatic activity loss. To minimize freeze-thaw effects, aliquot 150 &#181;L of the solution into sterile microcentrifuge tubes prior to initial freezing, and thaw individual aliquots as needed for experiments.</li>
	<li>2% (vol/vol) Fetal Bovine Serum (FBS) in Phosphate-Buffered Saline (PBS): Prepare 1&#215; PBS by mixing 5 mL of 10&#215; PBS with 45 mL of UltraPure DNase/RNase-free dH2O. Add 1 mL of FBS to 49 mL of 1&#215; PBS and mix thoroughly. 
	NOTE: Prepare this solution freshly during Step 2.1.2. Allocate 15 mL per tissue sample.</li>
	<li>Collagenase and dispase (CD) enzyme stock solution: Dissolve 100 mg of Dispase and 100 mg of Collagenase separately in 1 mL of UltraPure DNase/RNase-free dH2O each. Combine solutions to obtain a 2 mL final volume (100 mg/mL enzyme concentration). 
	NOTE: Aliquot 500 &#181;L into sterile microcentrifuge tubes and store at -20 &#176;C. Vortex before use to avoid activity loss from freeze-thaw cycles. It is important to note that the powder form can cause severe skin/eye irritation and respiratory distress. Always handle it in a chemical fume hood while wearing nitrile gloves and an N95 mask. In case of exposure, immediately irrigate the affected area with copious water and seek medical attention.</li>
	<li>CD enzyme working solution: Mix 1.5 mL of CD enzyme stock solution with 43.5 mL of 2% (vol/vol) FBS in PBS (from Step 1.3) by gentle inversion. 
	NOTE: Prepare immediately before tissue dissociation (Step 2.3.4). This volume is optimized for three mice; adjust proportionally as needed.</li>
	<li>22% (vol/vol) Percoll solution: Combine 11 mL of Percoll, 5 mL of 10&#215; PBS, and 34 mL of UltraPure DNase/RNase-free dH2O to prepare a 50 mL solution. 
	NOTE: Filter-sterilize through a 0.22 &#181;m PES membrane. It can be stored at 4 &#176;C for one month without significant loss of stability.</li>
	<li>Mechanical dissociation buffer: Add 1 mL of DNase I (10 mg/mL) to 10 mL of 2% (vol/vol) FBS in PBS (from Step 1.3). Mix by pipetting.</li>
	<li>BSA solutions: Prepare 1%, 20%, and 35% (wt/vol) BSA solutions separately as follows:
	<ol>
		<li>1% (wt/vol) BSA: Dissolve 0.15 g of BSA in 15 mL of Dulbecco&#39;s Phosphate Buffered Saline (DPBS).</li>
		<li>20% (wt/vol) BSA: Dissolve 3 g of BSA in 15 mL of DPBS.</li>
		<li>35% (wt/vol) BSA: Dissolve 0.7 g of BSA in 2 mL of DPBS. 
		NOTE: Filter all solutions through a 0.22 &#181;m PES membrane before use.</li>
	</ol>
	</li>
	<li>Hanks&#39; Balanced Salt Solution (HBSS)/BSA/Glucose Buffer: Add 167 &#181;L of 30% (wt/vol) glucose (from Step 1.1) and 1.4 mL of 35% (wt/vol) BSA (from Step 1.8) to 50 mL of HBSS, mix gently. 
	NOTE: Prepare this solution freshly under sterile conditions during Step 2.1.2. Store at 4 &#176;C until use.</li>
	<li>Tissue Collection Buffer: Supplement 12 mL of HBSS/BSA/Glucose Buffer (from Step 1.9) with 800 &#181;L of DNase I (10 mg/mL). Use for temporary tissue storage and pipette rinsing. 
	NOTE: Prepare this solution freshly under sterile conditions during Step 2.1.2.</li>
	<li>1.25% (w/v) Tribromoethanol Solution: Dissolve 1.25 g of 2,2,2-Tribromoethanol in 2.5 ml of 2-Methyl-2-butanol , then add this mixture to 97.5 ml of sterile ddH2O or physiological saline to obtain a clear 1.25% Tribromoethanol Solution. 
	NOTE: Prepare the solution fresh before each use. Store it protected from light at room temperature and use it promptly.</li>
</ol>
2. Experimental Procedure (using three mice as an example)
<ol>
	<li>Pre-experiment preparation (Timing: 30 min)
	<ol>
		<li>Autoclave required consumables and disinfect surgical instruments with 75% (vol/vol) ethanol.</li>
		<li>Prepare working solutions: 1&#215; PBS (from Step 1.3), 2% (vol/vol) FBS in PBS (from Step 1.3), HBSS/BSA/glucose solution (from Step 1.9), and tissue collection buffer (from Step 1.10).</li>
		<li>Pour 10 mL of 1&#215; PBS into a 10 cm cell cluture dish and keep it on ice.</li>
		<li>Prepare three ampoules, add 3 mL of tissue collection buffer to each, and keep them on ice.</li>
		<li>Prepare three 15 mL centrifuge tubes, add 10 mL of 2% (vol/vol) FBS in PBS to each, and keep them on ice.</li>
	</ol>
	</li>
	<li>Mouse brain harvesting (Timing: 30 min)
	<ol>
		<li>Mice were deeply anesthetized by intraperitoneal injection of tribromoethanol (from Step 1.11) at a dose of 0.2 mL per 10 g body weight. After the loss of pedal reflexes, euthanasia was performed by cervical dislocation. 
		NOTE: If the mouse did not reach a surgical plane of anesthesia (assessed by the absence of a pedal withdrawal reflex) within 5-10 minutes after injection, a supplemental intraperitoneal dose of tribromoethanol, equal to one-third of the initial dose, was administered.</li>
		<li>Spray the head/neck region with 75% ethanol. Make a midline scalp incision from occipital to frontal regions to expose the skull. Perform a transverse incision anterior to the bilateral orbits, followed by lateral incisions along the inferior margins of the cerebellum to detach the skull base. Finally, incise the calvarium longitudinally along the sagittal suture from posterior to anterior. Lift the cranial flap to expose the dura-enclosed brain. Gently elevate the brain while severing the optic nerves and the medulla oblongata-spinal cord connection with surgical scissors15. Place the intact brain in a 10 cm dish containing 10 mL of ice-cold 1&#215; PBS. 
		NOTE: Handle tissue gently to avoid damage.</li>
		<li>Transfer the brain to aluminum foil. Using forceps, lift the dura mater from the margins and excise it along the midline sulci. After complete dura removal, expose the subarachnoid space. Rinse the subarachnoid space with HBSS solution, then dissect vessels using a blunt needle and carefully remove the pia mater16. Section the brain coronally into 2-3 mm thick slices using a razor blade. Discard the olfactory bulb and caudal-most sections. Isolate the cerebral cortex along the corpus callosum and transfer cortical tissue to ampoules containing 3 mL of ice-cold tissue collection buffer11.</li>
		<li>Pericyte isolation in this protocol specifically targets the cerebral cortex. For other regions of interest: Place coronal sections in a two-well slide with one drop of 2% (vol/vol) FBS in PBS. Identify target regions under dissection microscopy, isolate manually, and proceed with identical subsequent processing steps.</li>
	</ol>
	</li>
	<li>Tissue dissociation (Timing: 3-3.5 h)
	<ol>
		<li>Mince tissues in ampoules using surgical scalpels into fragments of approximately 1 mm &#215; 1 mm. 
		NOTE: Complete mincing within 5 min to preserve tissue freshness.</li>
		<li>Pre-rinse pipette tips with tissue collection buffer. Slowly aspirate minced fragments toward the tip apex while maintaining vertical orientation (tip downward). Gently expel fragments into pre-chilled 15 mL centrifuge tubes (prepared in Step 2.1.5). 
		NOTE: Avoid aspirating fragments into the upper barrel to prevent adherence to the pipette wall.</li>
		<li>Wash fragment-containing ampoules (from Step 2.3.1) with 2% (vol/vol) FBS in PBS and transfer the wash to centrifuge tubes. Centrifuge at 300 x g at 4 &#176;C for 5 min.</li>
		<li>Discard supernatants post-centrifugation. Resuspend pellets in 3 mL of CD enzyme working solution (from Step 1.5) per tube. Aliquot each suspension equally into three tubes (total 9 tubes, 1 mL/tube), then supplement with 4 mL of CD enzyme working solution (final volume: 5 mL/tube).</li>
		<li>Incubate tubes in a 37 &#176;C hybridization oven for 100 min with orbital shaking at 100 x g14.</li>
		<li>Prepare tissue dissociation solution during incubation by adding 1 mL of DNase I solution to 10 mL of 2% (vol/vol) FBS in PBS (from Step 1.10).</li>
		<li>Terminate digestion by centrifugation at 300 x g at 4 &#176;C for 5 min. 
		NOTE: Assess digestion efficiency microscopically using a 10-20 &#181;L aliquot. The ideal endpoint manifests as bead-like vascular fragments; elongated vascular segments indicate under-digestion, whereas predominant single cells signify over-digestion.</li>
		<li>Resuspend pellets in 1 mL of tissue dissociation solution per tube. Dissociate mechanically by pipetting 100 times11. 
		NOTE: Avoid full pipette volume aspiration to minimize bubble formation and preserve cell viability.</li>
		<li>Pool all suspensions (from a total of 9 digestion tubes representing the brain tissues of 3 mice) into a 50 mL centrifuge tube. Rinse each digestion tube with 2 mL of 2% (vol/vol) FBS in PBS and combine washes. Centrifuge at 300 x g at 4 &#176;C for 5 min.</li>
		<li>After centrifugation, distinct layers are visible. Carefully discard the uppermost layer (containing impurities and small cellular debris), while retaining the intermediate layer (consisting of cells or cell clusters with lower density) and the bottom layer (intact cells and partially undigested tissue fragments). Add 12.5 mL of 20% BSA solution and gently invert the tube to resuspend the pellets. Centrifuge again at 1000 &#215; g at 4 &#176;C for 20 min.</li>
		<li>Collect the bottom pellet (enriched in intact cells with higher purity) and transfer the supernatant (primarily containing myelin debris, lipids, and a small number of cells) into a new 50 mL centrifuge tube. Resuspend by gentle inversion and repeat centrifuge at 1,000 x g at 4 &#176;C for 20 min.</li>
		<li>Resuspend each pellet obtained from the two centrifugation steps (step 2.3.10 and Step 2.3.11) in 2 mL of 2% (vol/vol) FBS in PBS each (final combined volume: 4 mL). 
		NOTE: Avoid touching the tube walls during resuspension to minimize contamination by debris.</li>
		<li>Combine the two resuspended suspensions from the previous step into a 15 mL centrifuge tube. Centrifuge at 300 &#215; g, 4 &#176;C for 5 min. Discard the supernatant (waste buffer containing BSA, cell debris, and potentially trace amounts of residual myelin lipids). Resuspend the pellet (the final purified and washed, high-purity intact target cells) in 2 mL of PBS solution containing 2% FBS. 
		NOTE: This step is to transfer the cells into a clean, suitable suspension buffer for downstream applications.</li>
		<li>Prepare four 15 mL centrifuge tubes, each containing 4 mL of 22% Percoll solution. Gently layer 0.5 mL aliquots of the resuspension onto the Percoll gradient (four aliquots total) to maximize cell yield. Centrifuge at 560 x g at 4 &#176;C for 10 min. 
		NOTE: Set the centrifuge to &#34;no brake&#34; to avoid vortex-induced disruption of the density gradient, ensuring myelin debris remains stabilized in the supernatant.</li>
		<li>Carefully aspirate supernatants. Resuspend pellets in 1 mL of HBSS/BSA/glucose buffer per tube. Pool suspensions into a new 15 mL tube. Rinse Percoll tubes with 2 mL of HBSS/BSA/glucose buffer and combine washes. Centrifuge at 300 x g at 4 &#176;C for 5 min).</li>
	</ol>
	</li>
	<li>Flow cytometry sorting preparation and acquisition (Timing: 50 min)
	<ol>
		<li>Carefully aspirate the supernatant. Resuspend the cell pellet in 1 mL of HBSS/BSA/glucose buffer.</li>
		<li>Add 5 &#181;L of Fc receptor (FcR) blocking reagent to the cell suspension and incubate at 4 &#176;C for 5 min.</li>
		<li>Add the following fluorescently conjugated antibodies to the cell suspension at the indicated volumes (sufficient for tissue derived from 3 mice): 5 &#181;L of CD45-PE; 5 &#181;L of CD41-PE; 20 &#181;L of CD31-APC; 15 &#181;L of CD13-FITC.</li>
		<li>Incubate the cells on ice for 20 min in the dark. 
		NOTE: Protect from light. Avoid stirring.</li>
		<li>Add 6 mL of HBSS/BSA/glucose buffer to wash the cells. Centrifuge at 300 x g at 4 &#176;C for 5 min.</li>
		<li>Aspirate the supernatant. Resuspend the cell pellet in 1 mL of HBSS/BSA/glucose buffer. Add 200 &#181;L of 7-aminoactinomycin D (7-AAD) solution (to exclude dead cells) and incubate on ice for 10 min. Subsequently, adjust the volume to 2 mL with HBSS/BSA/glucose buffer. 
		NOTE: Protect from light.</li>
		<li>Add 1% BSA solution to sorting collection tubes (15 mL conical tubes), gently swirl to rinse the inner surfaces. Aspirate excess solution, leaving exactly 1 mL of 1% BSA solution in each tube to receive sorted cells. Store temporarily on ice.</li>
		<li>Filter stained cell suspension through a 40 &#181;m strainer immediately before sorting.</li>
		<li>Establish an initial gate based on forward scatter (FSC-A) and side scatter (SSC-A) parameters to exclude cellular debris exhibiting low FSC-A/SSC-A signals.</li>
		<li>Depletion of hematopoietic lineage cells was achieved through dual-negative gating for CD45 and CD41, effectively eliminating contamination from leukocytes, immune cells, and platelets.</li>
		<li>Capture putative pericytes by CD13-positive selection from vascular wall cells.</li>
		<li>Evaluate membrane integrity via 7-AAD fluorescent probing; exclude apoptotic cells by 7-AAD&#8315; gating. 
		NOTE: Collect high-purity CD31&#8315;/CD13&#8314; pericytes through CD31-negative selection. Collect the sorted pericytes directly into tubes containing 1% BSA (from Step 2.4.7) and place them on ice. Immediately prepare single-cell suspensions for counting. Then load the samples directly for sequencing without any intermediate culture steps.</li>
	</ol>
	</li>
	<li>scRNA seq and Analysis (Tming: 3-4 days)
	<ol>
		<li>Single-cell suspensions of sorted pericytes were prepared following the protocol from the 10x Genomics &#34;GEM-X Universal 3&#39; Gene Expression v4 4-plex Reagent Kits User Guide&#34; (CG000731)17,18. The cell suspension prepared for loading had a viability of &#62; 90% and was diluted to a concentration within the recommended range of 300-2000 cells/&#181;L.</li>
		<li>Combine single-cell suspension with barcoded gel beads and enzymes, then partition into Gel Beads-in-Emulsion (GEMs) via microfluidic droplet encapsulation19. Perform in situ cell lysis, reverse transcription, and barcode-cDNA linkage.</li>
		<li>Break GEMs to recover products. Amplify cDNA by PCR using the following protocol: initial denaturation at 9 &#176;C for 4 s; followed by cycling (13 cycles for &#60;500 target cells, 12 cycles for 501-6000 target cells) consisting of denaturation at 9 &#176;C for 2 s, annealing at 9 &#176;C for 3 s, and extension at 7 &#176;C for 6 s per cycle; and a final extension at 72 &#176;C for 1 min followed by a hold at&#160;4 &#176;C. Validate the amplified products for fragment size (200-300 bp) and yield. 
		NOTE: The amplification products can be stored at 4 &#176;C for up to 72 h or at -2 &#176;C for one week, or they can be processed directly in the next step.</li>
		<li>For qualified cDNA: Fragment, perform end repair, ligate P7 adapter, and amplify with index primers. Size-select fragments to construct sequencing libraries.</li>
		<li>Sequence libraries on the Illumina NovaSeq platform following QC validation.</li>
		<li>Demultiplex sequencing data to extract cell barcodes, unique molecular identifier (UMIs), and RNA sequences. Align reads to the reference genome.</li>
		<li>Quantify UMI counts, estimate valid cell numbers, and generate a gene-barcode expression matrix20.</li>
		<li>Remove doublets using the DoubletFinder package (v2.0.6) in R. Predict based on artificial nearest-neighbor identification to ensure data quality.</li>
		<li>Conduct principal component analysis (PCA) and Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction using Seurat (v5.0). Identify cell clusters and visualize pericyte-specific marker genes to validate cellular identity.</li>
	</ol>
	</li>
</ol>

<ol><li>Payne, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pericyte+microenvironment+during+vascular+development%5BTitle%5D)+AND+%22Microcirculation%22%5BJournal%5D)">The pericyte microenvironment during vascular development.</a> Microcirculation. 26 (8), e12554(2019).</li>
<li>Ma, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blood-brain+barrier-associated+pericytes+internalize+and+clear+aggregated+amyloid-%CE%B242+by+LRP1-dependent+apolipoprotein+E+isoform-specific+mechanism%5BTitle%5D)+AND+%22Mol+Neurodegener%22%5BJournal%5D)">Blood-brain barrier-associated pericytes internalize and clear aggregated amyloid-β42 by LRP1-dependent apolipoprotein E isoform-specific mechanism.</a> Mol Neurodegener. 13 (1), 57(2018).</li>
<li>Mäe, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+analysis+of+blood-brain+barrier+response+to+pericyte+loss%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Single-cell analysis of blood-brain barrier response to pericyte loss.</a> Circ Res. 128 (4), e46-e62 (2021).</li>
<li>Li, P., Fan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pericyte+loss+in+diseases%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">Pericyte loss in diseases.</a> Cells. 12 (15), 1931(2023).</li>
<li>Oliveira, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cultured+brain+pericytes+adopt+an+immature+phenotype+and+require+endothelial+cells+for+expression+of+canonical+markers+and+ECM+genes%5BTitle%5D)+AND+%22Front+Cell+Neurosci%22%5BJournal%5D)">Cultured brain pericytes adopt an immature phenotype and require endothelial cells for expression of canonical markers and ECM genes.</a> Front Cell Neurosci. 17, 1165887(2023).</li>
<li>Yao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Challenges+in+pericyte+research%3Apericyte-specific+and+subtype-specific+markers%5BTitle%5D)+AND+%22Transl+Stroke+Res%22%5BJournal%5D)">Challenges in pericyte research:pericyte-specific and subtype-specific markers.</a> Transl Stroke Res. 13 (6), 863-865 (2022).</li>
<li>Vanlandewijck, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+molecular+atlas+of+cell+types+and+zonation+in+the+brain+vasculature%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A molecular atlas of cell types and zonation in the brain vasculature.</a> Nature. 554 (7693), 475-480 (2018).</li>
<li>Alliot, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pericytes+and+periendothelial+cells+of+brain+parenchyma+vessels+co-express+aminopeptidase+N%2C+aminopeptidase+A%2C+and+nestin%5BTitle%5D)+AND+%22J+Neurosci+Res%22%5BJournal%5D)">Pericytes and periendothelial cells of brain parenchyma vessels co-express aminopeptidase N, aminopeptidase A, and nestin.</a> J Neurosci Res. 58 (3), 367-378 (1999).</li>
<li>Huang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation%2C+culture%2C+and+characterization+of+primary+endothelial+cells+and+pericytes+from+mouse+sciatic+nerve%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">Isolation, culture, and characterization of primary endothelial cells and pericytes from mouse sciatic nerve.</a> J Neurosci Methods. 416, 110366(2025).</li>
<li>Armulik, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pericytes+regulate+the+blood-brain+barrier%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Pericytes regulate the blood-brain barrier.</a> Nature. 468 (7323), 557-561 (2010).</li>
<li>Crouch, E. E., Doetsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FACS+isolation+of+endothelial+cells+and+pericytes+from+mouse+brain+microregions%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">FACS isolation of endothelial cells and pericytes from mouse brain microregions.</a> Nat Protoc. 13 (4), 738-751 (2018).</li>
<li>Eichhorn, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+tissue+factor+and+platelet%2Fleukocyte+markers+on+extracellular+vesicles+reflect+platelet-leukocyte+interaction+in+severe+COVID-19%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Expression of tissue factor and platelet/leukocyte markers on extracellular vesicles reflect platelet-leukocyte interaction in severe COVID-19.</a> Int J Mol Sci. 24 (23), 16886(2023).</li>
<li>Smyth, L. C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Markers+for+human+brain+pericytes+and+smooth+muscle+cells%5BTitle%5D)+AND+%22J+Chem+Neuroanat%22%5BJournal%5D)">Markers for human brain pericytes and smooth muscle cells.</a> J Chem Neuroanat. 92, 48-60 (2018).</li>
<li>Wu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epitranscriptomic+mechanisms+of+N6-methyladenosine+methylation+regulating+mammalian+hypertension+development+by+determined+spontaneously+hypertensive+rats+pericytes%5BTitle%5D)+AND+%22Epigenomics%22%5BJournal%5D)">Epitranscriptomic mechanisms of N6-methyladenosine methylation regulating mammalian hypertension development by determined spontaneously hypertensive rats pericytes.</a> Epigenomics. 11 (12), 1359-1370 (2019).</li>
<li>Mirzadeh, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+subventricular+zone+en-face%3Awholemount+staining+and+ependymal+flow%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">The subventricular zone en-face:wholemount staining and ependymal flow.</a> J. Vis. Exp. (39), e1938(2010).</li>
<li>Zappaterra, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+cerebrospinal+fluid+from+rodent+embryos+for+use+with+dissected+cerebral+cortical+explants%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Isolation of cerebrospinal fluid from rodent embryos for use with dissected cerebral cortical explants.</a> J. Vis. Exp. (73), e50333(2013).</li>
<li>Zou, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single-cell+transcriptomic+atlas+of+human+skin+aging%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">A single-cell transcriptomic atlas of human skin aging.</a> Dev Cell. 56 (3), 383-397.e8 (2021).</li>
<li>Bahl, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+deep+learning+to+quantify+neuronal+activation+from+single-cell+and+spatial+transcriptomic+data%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Using deep learning to quantify neuronal activation from single-cell and spatial transcriptomic data.</a> Nat Commun. 15 (1), 779(2024).</li>
<li>Klein, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Droplet+barcoding+for+single-cell+transcriptomics+applied+to+embryonic+stem+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells.</a> Cell. 161 (5), 1187-1201 (2015).</li>
<li>Zheng, G. X. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Massively+parallel+digital+transcriptional+profiling+of+single+cells%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Massively parallel digital transcriptional profiling of single cells.</a> Nat Commun. 8 (1), 14049(2017).</li>
<li>Song, H. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptomic+comparison+of+human+and+mouse+brain+microvessels%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Transcriptomic comparison of human and mouse brain microvessels.</a> Sci Rep. 10 (1), 12358(2020).</li>
<li>He, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+the+brain+mural+cell+transcriptome%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Analysis of the brain mural cell transcriptome.</a> Sci Rep. 6, 35108(2016).</li>
<li>Stebbins, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+pluripotent+stem+cell-derived+brain+pericyte-like+cells+induce+blood-brain+barrier+properties%5BTitle%5D)+AND+%22Sci+Adv%22%5BJournal%5D)">Human pluripotent stem cell-derived brain pericyte-like cells induce blood-brain barrier properties.</a> Sci Adv. 5 (3), eaau7375(2019).</li>
<li>Crouch, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regional+and+stage-specific+effects+of+prospectively+purified+vascular+cells+on+the+adult+V-SVZ+neural+stem+cell+lineage%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Regional and stage-specific effects of prospectively purified vascular cells on the adult V-SVZ neural stem cell lineage.</a> J Neurosci. 35 (11), 4528-4539 (2015).</li>
<li>Ruan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Screening+and+identification+of+muscle+pericyte+selective+markers%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Screening and identification of muscle pericyte selective markers.</a> Sci Rep. 15 (1), 28874(2025).</li>
<li>Dias, D. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pericyte-derived+fibrotic+scarring+is+conserved+across+diverse+central+nervous+system+lesions%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Pericyte-derived fibrotic scarring is conserved across diverse central nervous system lesions.</a> Nat Commun. 12 (1), 5501(2021).</li>
<li>Thalgott, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-invasive+characterization+of+pericyte+dysfunction+in+mouse+brain+using+functional+ultrasound+localization+microscopy%5BTitle%5D)+AND+%22Nat+Biomed+Eng%22%5BJournal%5D)">Non-invasive characterization of pericyte dysfunction in mouse brain using functional ultrasound localization microscopy.</a> Nat Biomed Eng. , (2025).</li>
<li>Grant, R. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organizational+hierarchy+and+structural+diversity+of+microvascular+pericytes+in+adult+mouse+cortex%5BTitle%5D)+AND+%22J+Cereb+Blood+Flow+Metab%22%5BJournal%5D)">Organizational hierarchy and structural diversity of microvascular pericytes in adult mouse cortex.</a> J Cereb Blood Flow Metab. 39 (3), 411-425 (2019).</li>
<li>O'flanagan, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissociation+of+solid+tumor+tissues+with+cold+active+protease+for+single-cell+RNA-seq+minimizes+conserved+collagenase-associated+stress+responses%5BTitle%5D)+AND+%22Genome+Biol%22%5BJournal%5D)">Dissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses.</a> Genome Biol. 20 (1), 210(2019).</li>
</ol>

The authors declare no conflicts of interest.

This study describes a systematically optimized and validated FACS strategy based on CD13&#8314;/CD31&#8315; multicolor labeling for the highly efficient isolation of mouse brain vascular pericytes24. Flow cytometric and subsequent scRNA-seq analyses confirmed that the method precisely discriminates pericytes from endothelial cells and yields cells that consistently meet all critical quality control metrics for scRNA-seq, including viability, aggregation rate, live cell concentration, and nucleate...

Pericytes are an important type of cell located along microvessels, typically embedded within the capillary basement membrane and enveloping endothelial cells1. In the brain, pericytes, together with endothelial cells, astrocytes, and other cell types, form the neurovascular unit and are integral to the structure and function of the blood-brain barrier (BBB). Pericytes play essential roles in the regulation of cerebral blood flow, vascular remodeling, and maintenance of vascular homeostasis. They maintain the integrity of the BBB through close physical interactions and paracrine signaling with neighboring cells2; dynamically regulate transendothelial transport via endocytosis and the expression of specific transporter proteins; and contribute to cerebral hemodynamic stability through their contractile and relaxant activities3. Dysfunction of pericytes, as well as alterations in their coverage or density, can lead to vascular dysregulation and neurological deficits, ultimately resulting in pathological changes associated with various neurological disorders4. However, the inherent heterogeneity of pericytes poses significant challenges for further mechanistic studies.
With the advancement of single-cell RNA sequencing (scRNA-seq) technology, an increasing number of studies have utilized scRNA-seq to explore cellular heterogeneity and identify key targets in the pathogenesis and progression of diseases. Commonly used pericyte markers include platelet-derived growth factor receptor-beta (PDGFR-β)5, neuron-glial antigen 2 (NG2), and alpha-smooth muscle actin (α-SMA). However, these markers have relatively poor specificity6, as they can simultaneously label other vascular cells, such as smooth muscle cells, and neural precursor cells like oligodendrocyte progenitor cells, making it difficult to distinguish pericytes from other brain cells. In search of a more robust alternative, we turned to CD13 (Aminopeptidase N, APN), a marker widely recognized for its strong and consistent expression in cerebrovascular pericytes in previous studies7,8,9,10. In our study, we conducted immunofluorescence staining on brain tissue sections, and the results confirmed CD13 expression in pericytes, demonstrating its effectiveness in distinguishing pericytes from endothelial cells, as the CD13 signal was exclusively associated with perivascular cells and absent from CD31⁺ endothelial cells. (Figure 1A). However, it is important to note that CD13 expression is not entirely exclusive to pericytes; detectable levels are also observed in smooth muscle cells of arterioles and venules (Figure 1B,C).
<img alt="Fluorescence microscopy of α-SMA, CD13, CD31 in cells; DAPI-stained nuclei; cellular marker study." class="xfigimg" src="/files/ftp_upload/69749/69749fig1.jpg" data-altupdatedat="1785816036000" data-alt="Figure 1"> 
Figure 1: Immunofluorescence staining shows CD13 expression in brain vascular pericytes and smooth muscle cells, with no detectable signal in endothelial cells. (A) Representative immunofluorescence imaging of the arteriole-capillary transition zone in the brain microvasculature. The merged image shows CD13⁺ pericyte (green) ensheathing CD31⁺ endothelial cells (red). α-SMA+ smooth muscle cells (cyan) are localized to the arteriolar region and are absent from capillaries. (B) Cross-section of an arteriole. The images demonstrate clear co-localization of CD13⁺ pericyte (green) and α-SMA+ smooth muscle cells (cyan) in the surrounding smooth muscle layer, confirming CD13 expression in arteriolar smooth muscle cells. (C) Cross-section of a venule. Similarly, CD13 (green) is detectable in the α-SMA smooth muscle cells (arrowheads) of the venular wall. Scale bar: 20 µm (A-C). <a href="https://www.jove.com/files/ftp_upload/69749/69749fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To obtain mouse cortical vascular pericytes that meet the requirements for scRNA-seq, we performed fluorescence-activated cell sorting (FACS) using CD13⁺/CD31⁻ antibody labeling for pericyte isolation11. Initially, CD45 and CD41 markers were used to exclude hematopoietic cells from brain tissue suspensions12. Subsequently, pericytes were enriched by selecting for CD13+ cells, and endothelial cells were further excluded by gating for CD31- populations, thereby isolating the target pericyte population13.
We acknowledge that the CD13-positive selection step concomitantly captures CD13-expressing smooth muscle cells from both arterioles and venules. To fulfill the critical requirements for scRNA-seq and maximize pericyte retention, we strategically selected this marker combination to prioritize pericyte yield at the sorting stage. Although this approach leads to the co-isolation of a minor population of smooth muscle cells, it effectively maximizes the recovery of all potential pericytes during the preliminary enrichment phase. This stepwise gating strategy significantly minimizes initial cell loss. In addition, to maximize pericyte viability and yield, we also employed a 20% BSA solution for post-dissociation cell resuspension and filtration, effectively removing debris while preserving sorted cell viability and purity14.
Using this method, we successfully distinguished brain vascular pericytes from other cell types, achieving high-purity and high-viability pericyte preparations that fulfill the stringent requirements for scRNA-seq. This technique provides a robust foundation for subsequent in-depth investigations into the roles of pericytes in various disease contexts.

<table><tbody><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibicol</td><td>A5669701</td><td>Aliquot and store at -20&amp;deg;C; can be stored at 4&amp;deg;C for up to 1 week after thawing</td></tr><tr><td>Phosphate Buffered Saline(10&amp;times;PBS, pH 7.4)</td><td>Life Technologies</td><td>70011-044</td><td>Dilute before use for cell washing</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7021-1KG</td><td>For preparing cell energy buffer</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A1933-25g</td><td>Protects cells and prevents adhesion</td></tr><tr><td>Dulbecco&#039;s&amp;nbsp; Phosphate Buffered Saline (DPBS,1&amp;times;)</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS,1&amp;times;)</td><td>Life Technologies</td><td>14175-095</td><td>For cell washing and resuspension</td></tr><tr><td>UltraPure DNase/RNase-Free Distilled Water</td><td>Life Technologies</td><td>10977-015</td><td>For preparing nuclease-free reaction systems</td></tr><tr><td>Collagenase/Dispase</td><td>Sigma-Aldrich</td><td>11097113001-100mg</td><td>For tissue digestion and cell dissociation; aliquot and store at -20&amp;deg;C</td></tr><tr><td>DNaseI(RNase-Free)</td><td>TIANGEN</td><td>RT411-1500U</td><td>Prevents DNA aggregation; aliquot and store at -20&amp;deg;C</td></tr><tr><td>Percoll</td><td>Sigma-Aldrich</td><td>P1644-100ML</td><td>For cell separation; store at 4&amp;deg;C</td></tr><tr><td>FITC Rat Anti-Mouse CD13</td><td>BD Biosciences</td><td>558744</td><td>Fluorescent conjugate; store protected from light at 4&amp;deg;C for up to 1 month</td></tr><tr><td>APC Rat Anti-Mouse CD31</td><td>BD Biosciences</td><td>551262</td><td>Store protected from light at 4&amp;deg;C for several months</td></tr><tr><td>PE Rat Anti-Mouse CD41</td><td>BD Biosciences</td><td>558040</td><td>Store protected from light at 4&amp;deg;C for several months</td></tr><tr><td>PE Rat Anti-Mouse CD45</td><td>BD Biosciences</td><td>553081</td><td>Store protected from light at 4&amp;deg;C for several months</td></tr><tr><td>FITC Rat IgG1, &amp;kappa; Isotype Control</td><td>BD Biosciences</td><td>552916</td><td>Fluorescence control; store protected from light at 4&amp;deg;C</td></tr><tr><td>APC Rat IgG2a, &amp;kappa; Isotype Control</td><td>BD Biosciences</td><td>553932</td><td>Store protected from light at 4&amp;deg;C for several months</td></tr><tr><td>PE Rat IgG2b, &amp;kappa; Isotype Control</td><td>BD Biosciences</td><td>553989</td><td>Store protected from light at 4&amp;deg;C for several months</td></tr><tr><td>RNase AWAY Decontamination Reagent</td><td>Ambion</td><td>10328011</td><td>For eliminating RNase contamination</td></tr><tr><td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)</td><td>BD Pharmingen</td><td>553141</td><td>Prevent non-specific antibody binding</td></tr><tr><td>7-Aminoactinomycin D (7-AAD)</td><td>BD Pharmingen</td><td>559925</td><td>For dead cell exclusion.</td></tr><tr><td>2,2,2-Tribromoethanol</td><td>Sigma-Aldrich</td><td>T48402-25g</td><td>For the preparation of 1.25% (w/v) Tribromoethanol Solution</td></tr><tr><td>2-Methyl-2-butanol</td><td>Psaitong</td><td>M20008-500mL</td><td>For the preparation of 1.25% (w/v) Tribromoethanol Solution</td></tr><tr><td>C57BL/6 mouse</td><td>Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.</td><td>SYXK (Su) 2020-0041</td><td></td></tr><tr><td>50 mL centrifuge tube</td><td>Corning</td><td>430291</td><td></td></tr><tr><td>15 mL centrifuge tube</td><td>Corning</td><td>430791</td><td></td></tr><tr><td>cell cluture dish 100mm&amp;times;20mm</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>Cell Ranger</td><td></td><td>3.1.0/4.0.0</td><td>Single-Cell Sequencing Data Analysis Software</td></tr><tr><td>Loupe Cell Browser</td><td></td><td>4.1</td><td>Cell Browser</td></tr><tr><td>STAR</td><td></td><td>2.5.1b</td><td>RNA Alignment</td></tr><tr><td>irlbpy</td><td></td><td>-</td><td>Principal Component Analysis</td></tr><tr><td>tsne</td><td></td><td>0.15</td><td>Barnes-Hut t-SNE (t-Distributed Stochastic Neighbor Embedding) Analysis</td></tr><tr><td>k-means</td><td></td><td>0.17</td><td>K-Means Clustering</td></tr><tr><td>graph-based clustering</td><td></td><td>0.17</td><td>Graph-Based Clustering</td></tr><tr><td>FastQC</td><td></td><td>v0.11.2</td><td>Data Quality Control (QC)</td></tr></tbody></table>

isolation of pericytes from mouse cortical tissue using facs for single-cell sequencing

Isolation of Pericytes from Mouse Cortical Tissue Using FACS for Single-cell Sequencing

Pericytes, as a core component of the neurovascular unit, play a vital role in maintaining the integrity of the blood-brain barrier and regulating cerebral hemodynamic homeostasis through modulation of microvascular tone. Dysfunction of pericytes has been implicated in a variety of neurological disorders. To investigate the heterogeneity of pericytes and elucidate their mechanisms in neurovascular diseases at single-cell resolution, we established an optimized and reproducible protocol for isolating mouse brain pericytes suitable for single-cell RNA sequencing (scRNA-seq). This method builds upon the established CD13+/CD31- sorting strategy, but incorporates key enhancements tailored specifically for scRNA-seq applications. Briefly, brains were harvested from three adult C57BL/6 mice. The brain tissues were mechanically dissociated and subjected to enzymatic digestion to obtain single-cell suspensions. After sequential centrifugation, the final cell pellet was resuspended in 20% bovine serum albumin (BSA) solution, a pivotal step we introduced to minimize stress and aggregation, thereby maximizing viability and RNA quality for sequencing. This optimized FACS-based approach thus enables the robust isolation of viable mouse brain pericytes meeting the stringent requirements for scRNA-seq, offering a powerful tool for dissecting pericyte heterogeneity and their pathological roles in neurovascular diseases.

To achieve precise isolation of brain pericytes, cells dissociated from brain tissue were stained with PE Texas Red-conjugated anti-mouse CD41/CD45, FITC-conjugated anti-mouse CD13, and APC-conjugated anti-mouse CD31, establishing a multi-step sorting strategy integrating surface markers and viability detection (Figure 2). Initial gating based on forward scatter (FSC-A) and side scatter (SSC-A) parameters effectively excluded cellular debris (Figure 2, top-left ...

To obtain mouse brain cortical pericytes meeting the demands of scRNA-seq, we present a critically optimized FACS protocol based on CD13&#8314;/CD31&#8315; selection. Our key refinements significantly enhance cell viability and RNA integrity, enabling reliable transcriptomic profiling.

Pericytes, as a core component of the neurovascular unit, play a vital role in maintaining the integrity of the blood-brain barrier and regulating ...

Our research examine brain parasites focusing on their vascular roles and their interactions within the neurovascular unit to understand the cerebral function. Low imaging and the spatial FACS in a bowel process mapping or parasite function within brain vasculature revealing the location specific roles and therapeutic targets. To begin, spray the head and neck region of a euthanized mouse with 75%ethanol.Using a scalpel, make a midline scalp incision from the occipital to the frontal region to expose the skull. Perform a transverse incision anterior to the bilateral orbits, followed by lateral incisions along the inferior margins of the cerebellum to detach the skull base. Incise the calvarium longitudinally along the sagittal suture from posterior to anterior.Lift the cranial flap together with a duramater to expose the brain. Using surgical scissors, gently elevate the brain while severing the optic nerves and the medulla oblongata spinal cord connection. Place the intact brain gently into a 10 centimeter dish containing 10 milliliters of ice-cold PBS, avoiding any damage.Transfer the brain onto aluminum foil. Expose the subarachnoid space. After removing the dura with forceps, rinse it with Hank's balanced salt solution.Transfer the tissue to filter paper and remove the piamater by rolling. With a razor blade, cut and discard the olfactory bulb, and the caudal most sections, and section the brain coronally into slices two to three millimeters thick. Isolate the cerebral cortex along the corpus callosum.Transfer the cortical tissue into ampules containing three milliliters of ice cold tissue collection buffer. Using surgical scalpels, mince the tissue and ampules into fragments of approximately one by one millimeter, and complete the mincing within five minutes to preserve tissue freshness. Pre-rinse pipette tips with tissue collection buffer.Then slowly aspirate the minced fragments toward the tip apex while holding the pipette vertically. Gently expel the fragments into pre-chilled 15 milliliter centrifuge tubes. Wash the original ampules with 2%FBS in PBS saline.Transfer the wash into the same centrifuge tubes and centrifuge at 300G for five minutes at four degrees Celsius. Discard the supernatant after centrifugation. Re-suspend each pellet in three milliliters of CD enzyme working solution.Aliquot the suspension equally into three tubes, and add additional enzyme solution to reach a final volume of five milliliters per tube. Incubate the tubes in a hybridization oven at 37 degrees Celsius for 100 minutes with orbital shaking at 100G. During incubation, add one milliliter of dnase one solution to 10 milliliters of 2%FBS in PBS.Terminate digestion by centrifuging at 300G at four degrees Celsius for five minutes. Then re-suspend each pellet in one milliliter of tissue dissociation solution. Pipette the solution 100 times to aid mechanical dissociation.Next, pool all suspensions into a single 50 milliliter centrifuge tube. Rinse each tube with two milliliters of buffer and combine the washes. After centrifugation at 300G for five minutes at four degrees Celsius and discarding the uppermost layer, add 12.5 milliliters of 20%BSA and invert to mix.Centrifuge at 1000 G at four degrees Celsius for 20 minutes. Now collect the bottom pellet and transfer the supernatant into a new 50 milliliter centrifuge tube. Re-suspend gently before centrifuging again.Re-suspend each pellet in two milliliters of 2%FBS in PBS. Then combine both suspensions into a single tube. Centrifuge the combined suspension at 300G at four degrees Celsius for five minutes.After discarding the supernatant, re-suspend the final pellet in two milliliters of PBS with 2%FBS. Prepare four 15 milliliter centrifuge tubes containing four milliliters of 22%per call solution. Gently layer 0.5 milliliter aliquots of the cell suspension onto each gradient and centrifuge at 560G for 10 minutes at four degrees Celsius.After aspirating the supernatant, re-suspend each pellet in one milliliter of HBSS BSA glucose buffer. Then pull the suspensions into a new 15 milliliter tube. Rinse the percol tubes with two milliliters of buffer and combine the washes before centrifugation at 300G for five minutes at four degrees Celsius.For flow cytometry, re-suspend the pellet in one milliliter of HBSS BSA glucose buffer after aspirating the supernadant. Add five microliters of FC receptor blocking reagent and incubate at four degrees Celsius for five minutes. Then pipette fluorescently conjugated antibodies to the suspension before incubating.Wash the cells with six milliliters of HBSS BSA glucose buffer. Then centrifuge the suspension at 300G at four degrees Celsius for five minutes. After aspirating the supernatant, re-suspend the pellet in one milliliter of buffer.Add 200 microliters of seven amino actinomycin D solution and incubate on ice for 10 minutes. Then adjust the final volume to two milliliters with HBSS buffer supplemented with BSA and glucose. Rinse the inner surface of sorting collection tubes with 1%BSA solution.Aspirate out the excess solution, leaving exactly one milliliter of solution in each tube. Store the tubes on ice to receive sorted cells. Cells dissociated from brain tissue were sequentially gated by forward scatter area and side scatter area to exclude cellular debris during flow cytometry sorting.CD45 positive and CD41 positive dual positive events were excluded to remove hematopoietic cells and platelet contaminants. CD13 positive vascular wall cells were enriched. And apoptotic cells were excluded using seven amino actinomycin D negative gating, followed by CD31 negative selection to isolate parasites.Flow cytometry sorting showed that nucleated cells accounted for 59.8%of all events. CD13 positive cells accounted for 1%of all events. And the final CD13 positive CD31 negative parasite population accounted for 0.7%of all events.UMAP dimensionality reduction of the sorted cells revealed six distinct major clusters indicating cellular heterogeneity within the isolated population. Z-score normalized heat map analysis demonstrated that parasite marker genes were highly and predominantly expressed in clusters zero through three. Cluster proportion analysis showed that clusters zero through three constituted 87.1%of the total population.Whereas smooth muscle cell markers were enriched in cluster four at 10.14%and fibroblast markers were enriched in cluster five at 2.77%This protocol allows researchers to isolate high purity wearable parasites suitable for downstream single cell sequencing and functional analysis. The main challenge is the absence of highly specific parasite markers, complicated isolation, and increasing the risk of secular contamination. Future studies can focus on identifying parasite specific markers, enabling improved isolate, deeper functional characterization, and targeted therapeutic investigations.

isolation-pericytes-from-mouse-cortical-tissue-using-facs-for-single

Research

JoVE Journal

Biochemistry

This video demonstrates a method for isolating cerebral pericytes from a mouse brain. The isolated cells are cultured in pericyte-specific media to promote pericyte proliferation.

Isolating and Culturing Mouse Cerebral Pericytes

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

 Murine Dermal Fibroblast Isolation by FACS

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

Isolation and Purification of Murine Cardiac Pericytes

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.

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

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

A High Output Method to Isolate Cerebral Pericytes from Mouse

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

We present a method for isolating endothelial cells and nuclei from the lumen of mouse carotid arteries exposed to stable or disturbed flow conditions to perform single-cell omics experiments.

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Learn to isolate mouse brain pericytes using FACS with CD13+/CD31- sorting for single-cell RNA sequencing. This optimized protocol enables high-quality analysis of pericyte heterogeneity in neurovascular disease research.

Isolation of Pericytes from Mouse Cortical Tissue Using FACS for Single-cell Sequencing

Explore More Videos