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A flow cytometry-based protocol for syngenic isolation of neurovascular unit cells from mouse and human tissues

Nature Protocols volume 18, pages 1510–1542 (2023)Cite this article

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Abstract

The neurovascular unit (NVU), composed of endothelial cells, pericytes, juxtaposed astrocytes and microglia together with neurons, is essential for proper central nervous system functioning. The NVU critically regulates blood–brain barrier (BBB) function, which is impaired in several neurological diseases and is therefore a key therapeutic target. To understand the extent and cellular source of BBB dysfunction, simultaneous isolation and analysis of NVU cells is needed. Here, we describe a protocol for the EPAM-ia method, which is based on flow cytometry for simultaneous isolation and analysis of endothelial cells, pericytes, astrocytes and microglia. This method is based on differential processing of NVU cell types using enzymes, mechanical homogenization and filtration specific for each cell type followed by combining them for immunostaining and fluorescence-activated cell sorting. The gating strategy encompasses cell-type-specific and exclusion markers for contaminating cells to isolate the major NVU cell types. This protocol takes ~6 h for two sets of one or two animals. The isolation part requires experience in animal handling, fresh tissue processing and immunolabeling for flow cytometry. Sorted NVU cells can be used for downstream applications including transcriptomics, proteomics and cell culture. Multiple cell-type analyses using UpSet can then be applied to obtain robust targets from single or multiple NVU cell types in neurological diseases associated with BBB dysfunction. The EPAM-ia method is also amenable to isolation of several other cell types, including cancer cells and immune cells. This protocol is applicable to healthy and pathological tissue from mouse and human sources and to several cell types compared with similar protocols.

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Fig. 1: Overview of EPAM-ia method for the syngenic isolation of neurovascular unit cells from the adult mouse brain.
Fig. 2: Separation of vascular cells from glial cells using nylon mesh filtration for differential processing of NVU cells.
Fig. 3: Final EPAM-ia method gating strategy on single-brain and purity comparison with the control sample of murine stroke model.
Fig. 4: EPAM-ia method-based isolation of NVU and tumor cells applied to an animal model of brain metastasis.
Fig. 5: Representative FACS plots showing the gating strategy for the isolation of the NVU cells as well as immune and tumor cells in brain tissue from mouse glioblastoma model.
Fig. 6: Representative FACS plots showing the gating strategy for the isolation of NVU cells from human brain tissue using the EPAM-ia protocol.
Fig. 7: Analysis of NVU cells isolated with EPAM-ia method for cell-specific and multicellular targeting.
Fig. 8: Schematic for downstream handling and analyses post EPAM-ia method.

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Article 01 December 2022

Zixuan Zhao, Xinyi Chen, … Hanry Yu

Data availability

All the raw data and derived data of this manuscript are available from the corresponding author upon reasonable request. The transcriptomic datasets referred to in this manuscript have been uploaded to NCBI database GSE163752, and also to other servers (https://bioinformatics.mpi-bn.mpg.de/SGD_Stroke, http://vcg.github.io/upset/ followed by pasting the JSON file link https://raw.githubusercontent.com/SGD2020/mcao/master/mcao.json)

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Acknowledgements

The authors acknowledge the contributions of B. Yalcin, J. Macas and C. Czupalla with the FACS compensation and troubleshooting of the sorter and for inputs regarding the gating strategy. The authors thank S. Momma and I. Kur for neuronal staining. The authors would like to also thank L. Hansen and N. Lohfink for assistance with mice handling. The authors acknowledge SFB 1039 for funding D.S. and SFB TR23 to Y.R., K.H.P. and S.L., and GO-IN fellowship (291776) for funding Sy.G. and M.D.T., LI 911/5-1 to S.L., EU JUSTBRAIN consortium and Sphingonet consortium of LeducQ foundation for funding K.D. The authors acknowledge LOEWE CePTER consortium for funding Y.R., K.H.P., S.L. and K.D. The authors acknowledge the Edinger Institute for funding all the consumables and softwares with respect to FACS sorting and analysis.

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Author notes

  1. These authors contributed equally: Sylvaine Guérit, Kavi Devraj.

Authors and Affiliations

  1. Department of Neurology, Goethe University, Frankfurt, Germany

    Daniel Spitzer & Tim Pütz

  2. Edinger Institute (Institute of Neurology), Goethe University, Frankfurt, Germany

    Daniel Spitzer, Maryam I. Khel, Tim Pütz, Jenny Zinke, Xiaoxiong Jia, Kathleen Sommer, Katharina Filipski, Karl H. Plate, Patrick N. Harter, Stefan Liebner, Yvonne Reiss, Mariangela Di Tacchio, Sylvaine Guérit & Kavi Devraj

  3. The Molecular Imaging Center, Department of Biomedicine, University of Bergen, Bergen, Norway

    Frits Thorsen

  4. Department of Neurosurgery, University Medical Center Rostock, Rostock, Germany

    Thomas M. Freiman

  5. Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany

    Stefan Günther

  6. German Cancer Consortium (DKTK) Partner site Frankfurt/Mainz, Frankfurt, Germany

    Karl H. Plate, Patrick N. Harter & Yvonne Reiss

  7. Center for Personalized Translational Epilepsy Research (CePTER), Frankfurt, Germany

    Karl H. Plate, Patrick N. Harter, Stefan Liebner, Yvonne Reiss & Kavi Devraj

  8. German Cancer Research Center (DKFZ), Heidelberg, Germany

    Karl H. Plate, Patrick N. Harter & Yvonne Reiss

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Contributions

D.S., Sy.G., T.P. and K.D. performed tissue dissociation and FACS staining of WT healthy mice and human specimens; J.Z., X.J. and K.S. performed intracardial tumor cell injections and metastasis model characterization by immunohistochemistry; D.S., J.Z. and X.J. performed tissue dissociation and FACS staining for mouse metastasis model; Sy.G. and K.D. performed FACS operation and sorting; M.D.T., Sy.G., and K.D. analyzed and designed the FACS gating strategy with inputs from Y.R. and S.L.; D.S. and M.D.T. performed FACS analysis and prepared FACS plots; D.S., Sy.G., T.P. and M.I.K. performed qPCR analysis; St.G. performed the transcriptomic analysis. Sy.G. created the figures and performed statistics with assistance from K.D.; F.T. established the mouse melanoma model; T.M.F. operated the patients and performed the resection of the biopsies; K.F., K.H.P. and P.N.H. performed dissection and neuropathological analysis of human tissue; Sy.G., Y.R., S.L., M.D.T. and K.D. developed and improved the enzyme combinations and antigen selection. D.S., Sy.G. and K.D. wrote the manuscript with inputs from all the authors. K.D. and Sy.G. designed the overall protocol and also supervised the study.

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Correspondence to Kavi Devraj.

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Key references using this protocol

Spitzer, D. et al. Acta Neuropathol. 144, 305–337 (2022): https://doi.org/10.1007/s00401-022-02452-1

Guérit, S. et al. Prog. Neurobiol. 199, 101937 (2021): https://doi.org/10.1016/j.pneurobio.2020.101937

Devraj, G. et al. Acta Neuropathol. 140, 183–208 (2020): https://doi.org/10.1007/s00401-020-02174-2

Extended data

Extended Data Fig. 1 Assessment of papain concentration in Digestion Mix 1 of the EPAM-ia method on cell yield and viability.

a) Gating strategy for the isolation of endothelial cells, pericytes (mural cells), astrocytes, and microglia from mouse cerebrum digested according to the EPAM-ia protocol with 0.1% of papain in the Digestion Mix 1, representative experiment of n=3. Initial FSC/SSC dot plot show 1,000,000 events. Percentages refer to the proportion of cells in the previous parental gate. (b) Normalized cell events of the four major NVU cell types isolated using different papain concentration in the Digestion Mix 1 (with 0.025%, n=4; 0.05% n=5; 0.1% n=3;). (c) Normalized number of dead cells using different concentrations of papain in the Digestion Mix 1.

Extended Data Fig. 2 Assessment of alternative kit-based Digestion Mix 1 and debris removal solution on cell yield, viability, and purity of the isolated NVU cells.

(a) Schematic indicating with an asterisk the two steps during the cell suspension preparation for which kit based solutions are used. (b) Identical gating strategy as in the fully defined EPAM-ia method is applied to obtained microglia, astrocytes, endothelial cells and pericytes (mural cells) from the same brain tissue sample with the kit based Digestion mix 1 and debris removal steps. The FSC/SSC plot shows 1,000,000 events. Percentages refer to the proportion of cells in the previous parental gate. (c) Normalized cell events of endothelial cells (EC), pericytes (PC), astrocytes (AC) and microglia (MG) isolated by flow cytometry after tissue dissociation using commercially available solutions for the Digestion Mix 1 and the debris removal (n=5). (d) Normalized number of dead cells. (e-h) Purity of kit-based and EPAM-ia method sorted cells is assessed by qPCR targeting cell type-specific markers of endothelial cells (e, Cdh5, Slca1a1), pericytes (f, Kcnj8), astrocytes (g, Aqp4, Slc1a3), and microglia (h, Tmem119). If no amplification was detected, the ∆Ct value is set at 15 by default (n=4). One way ANOVA followed by Dunnett’s multiple comparison test. *, †, § and $ indicate comparison to EC, PC, AC and MG respectively. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001 and ns: not significant. Panels a,e–h adapted with permission from ref. 31, Springer.

Extended Data Fig. 3 Enrichment analysis of the isolated NVU cell types and the all negative population.

(a) Detailed transcripts reads for representative cell type markers genes of endothelial cells (EC), pericytes (PC), astrocytes (AC) and microglia (MG) in healthy single brain (n=1) or stroke contralateral hemisphere tissue (n=4, 3-4 mice/preparation). (b) Heatmap representing log2 expression of selected choroid plexus and neuronal cell-specific marker genes in endothelial cells, pericytes, astrocytes, microglia and all negative (AN) population isolated from a single mouse cerebrum (n=1). (c) FACS plots showing isolation of NeuN positive neurons by EPAM-ia method. Cells isolated by EPAM-ia method were preselected with ACSA2 magnetic beads followed by brief fixation (0.4% PFA) and permeabilization (0.1% Triton X-100) before immunostaining (n=2). Percentages refer to the proportion of cells in the previous parent gate. First SSC/FSC gate shows 200,000 events.

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Spitzer, D., Khel, M.I., Pütz, T. et al. A flow cytometry-based protocol for syngenic isolation of neurovascular unit cells from mouse and human tissues. Nat Protoc 18, 1510–1542 (2023). https://doi.org/10.1038/s41596-023-00805-y

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