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Jan 2018

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Papain-based Single Cell Isolation of Primary Murine Brain Endothelial Cells Using Flow Cytometry
利用流式细胞技术进行原代小鼠脑内皮细胞的基于木瓜蛋白酶的单细胞分离   

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Abstract

Brain endothelial cells (BECs) form the integral component of the blood-brain barrier (BBB) which separates the systemic milieu from the brain parenchyma and protects the brain from pathogens and circulating factors. In order to study BEC biology, it was of particular interest to establish a method that enables researchers to investigate and understand the underlying molecular mechanisms regulating their function during homeostasis, aging and disease. Furthermore, due to the heterogeneity of the cerebrovasculature and different vessel types that comprise the BBB, it is of particular interest to isolate primary BECs for single cell analysis from various subregions of the brain, such as the neurogenic and highly vascularized hippocampus and to enrich for specific vessel types. In the past, approaches to isolate endothelial cells were dependent on transgenic mice and often resulted in insufficiently pure cell populations and poor yield. This protocol describes a technique that allows single-cell isolation of highly pure brain endothelial cell populations using fluorescence activated cell sorting (FACS). Briefly, after perfusion and careful removal of the meninges, and dissection of the cortex/hippocampus, the brain tissue is mechanically homogenized and enzymatically digested resulting in a single cell suspension. Cells are stained with fluorochrome-conjugated antibodies identifying CD31+ brain endothelial cells, as well as CD45+CD11b+ myeloid cells for exclusion. Using flow cytometry, cell populations are separated and CD31+BECs are sorted in bulk into RNA later or as single cells directly into either RNA lysis buffer for single or bulk RNA-Seq analyses. The protocol does not require the expression of a transgene to label brain endothelial cells and thus, may be applied to any mouse model. In our hands, the protocol has been highly reproducible with an average yield of 1 x 105 cells isolated from an adult mouse cortex/hippocampus.

Keywords: Brain endothelial cells (脑内皮细胞), Blood-brain barrier (血脑屏障), Neuro-vascular unit (神经血管单元), Single cell isolation (单细胞分离), Cell sorting (细胞分选)

Background

The Blood-brain barrier (BBB) is a complex vascular structure that separates and protects the brain from systemic circulating factors and immune cells while allowing selective transport of critical nutrients. This highly specialized vasculature regulates selective transport of metabolites across the barrier. BECs also form a central component of the neurovascular unit (NVU), with close association and crosstalk with various cell types in the brain including neural precursor cells, microglia and the brain-resident immune cells. The BBB not only limits the passage of ions and other molecules such as glucose but also prevents the uncontrolled exchange of toxins, bacteria, viruses and cells between the blood and the brain parenchyma (Abbott et al., 2010). Nutrient-rich, oxygenated blood is pumped into the brain through cerebral arterial BECs (arteries and arterioles), which are protected and supported by smooth muscle cells (SMCs) that cover the endothelium and form a basement membrane layered by astrocytic end-feet of the brain parenchyma (Figure 1). The blood is transferred to highly specialized capillaries, which are comprised of BECs that form unique tight junctions and are wrapped by pericytes (Peric.) within the endothelial basement membrane, which is then covered by astrocytic end-feet. BBB capillaries are the site of controlled transport of fluids and solutes into the CNS. Immuno-surveillance and occasional extravasation of leukocytes (Leuk.) into the CNS parenchyma occurs at the level of postcapillary venous cells (venules and veins) the vascular segments into which blood flows after passing through the capillaries. Postcapillary Venules contain enlarged perivascular space between the endothelial and astrocytic basement membranes where occasional immune cells can reside (Banks, 2016; Engelhardt et al., 2017).

The outlined fluorescent activated cell sorting (FACS) single cell isolation method is based on previously described procedure for BEC isolation (Tam et al., 2012), that have been further developed and modified in context of a study aiming to determine molecular changes occurring in brain endothelial cells during aging and exposure to aged blood (Yousef et al., 2018). A schematic of the FACS procedure for BEC isolation and an example of cell gating are shown in Figure 2. Aging results in the upregulation of chronic inflammatory processes involving endothelial cell activation that contributes to reduced neural precursor cell activity and increased neuroinflammation in the aging brain (Yousef et al., 2018). The technique was successfully applied in a collaborative effort to investigating the effects of an aged systemic milieu on hippocampal neurogenesis and microglia activation (Yousef et al., 2018). The study highlights the role of the vascular cell adhesion molecule 1 (VCAM1) as a negative regulator of adult neurogenesis and inducer of microglial activity. VCAM1 is only expressed in a very small subpopulation of cells under baseline conditions and thus, was not sufficiently immunolabeled using conventional two-step antibody staining to perform FACS analysis. To that end, mice were stimulated systemically with Lipopolysaccharide (LPS) from Salmonella typhimurium to increase VCAM1 expression. The mice were then retro-orbitally injected with fluorescently labeled anti-mouse VCAM1, which resulted in a reliable detection of the VCAM1 positive brain endothelial cell subpopulation. The CD31+VCAM1+ BECs in LPS-stimulated mice could be used to set positive and negative gates in the flow sorter to quantitatively assess CD31+VCAM1+ expression in normal young and aged mice, or young mice treated with young or aged plasma and to isolate this rare subpopulation (Yousef et al., 2018; Figure 3). Additionally, VCAM1 is enriched in arterial and venous BECs and can be used to enrich and study these two vessel segmental populations (Vanlandewijck et al., 2018; Yousef et al., 2018).

In the last decade, several protocols for brain endothelial cell isolation have been employed, which tend to use transgenic endothelial cells labeled with fluorescent proteins such as GFP (Daneman et al., 2010; Vanlandewijck et al., 2018). Because these techniques are dependent on transgenic endothelial cell markers, they may not easily be applied to diseased or normal aged mouse models. This protocol allows the isolation of highly pure brain endothelial cell population from any mouse strain or murine animal model using papain-based enzymatic digestion using the commercial neural dissociation kit (MACS Miltenyi Biotec, Miltenyi).


Figure 1. Schematic of the blood-brain barrier. Nutrient-rich, oxygenated blood is pumped into the brain through cerebral arterial BECs (arteries and arterioles), which are protected and supported by smooth muscle cells (SMCs) that cover the endothelium and form a basement membrane layered by astrocytic end-feet of the brain parenchyma. The blood is transferred to highly specialized capillaries, which are comprised of BECs that form unique tight junctions and are wrapped by pericytes (Peric.) within the endothelial basement membrane, which is then covered by astrocytic end-feet. BBB capillaries are the site of controlled transport of fluids and solutes into the CNS. Immuno-surveillance and occasional extravasation of leukocytes (Leuk.) into the CNS parenchyma occurs at the level of postcapillary venous cells (venules and veins) the vascular segments into which blood flows after passing through the capillaries. Postcapillary Venules contain enlarged perivascular space between the endothelial and astrocytic basement membranes where occasional immune cells can reside (Banks, 2016; Engelhardt et al., 2017). (Adopt from Yousef et al. [2018] Figure 2a)


Figure 2. Mouse Brain endothelial cell isolation through flow cytometry. A. Schematic of flow sorting of CD31+CD45- BECs from mouse cortex and hippocampi. Each isolated RNA sample is a pool of BECs from 2 mouse brains. B. FACS gating strategy to isolate single BECs. PI+ dead cells were excluded. CD11b+ and CD45+ cells were gated to exclude monocytes/macrophages and microglia. CD31+Cd11b-CD45- cells were defined as the BEC population. (Adopt from Yousef et al. [2018] Supplemental Figure 1a-b)


Figure 3. Identification of VCAM1+CD31+ BECs through flow cytometry. Vcam1fl/fl Slco1c1-CreERT2+/- mice (Cre+) received tamoxifen (100 mg/kg; i.p.) once daily for 5 days. After a 3-day resting period, mice were treated with LPS for 16 h and 2 h before perfusion (1 mg/kg, i.p.) and fluorescently labeled anti-VCAM1 mAb (100 µg, r.o.) for 2 h prior to cell isolation and flow cytometry analysis. Gating plots of CD31+VCAM1+ cells isolated from Gating plots are of tamoxifen-treated, LPS stimulated aged (19-month-old) Slco1c1-CreERT2+/--Vcam1fl/fl (Cre+) and littermate control mice lacking a copy of the Cre gene (Cre-), injected with fluorescently-tagged DL488 anti-VCAM1 mAb or IgG-DL488 isotype control (r.o.) 2 h before sacrifice.

Materials and Reagents

  1. Pipette tips must be sterile and low retention
  2. Whatman paper: GE Healthcare Whatman Quantitative Filter Paper Grade 40 (Fisher Scientific, GE Healthcare, catalog number: 09-927-541)
  3. Thermo Scientific Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane (Thermo Fisher, catalog number: 569-0020)
  4. 15 ml and 50 ml tubes (Corning, catalog numbers: 430790 and 430828)
  5. 1.5 ml and 2.0 ml Eppendorf Tubes Protein Low-Binding (Eppendorf, catalog numbers: 022431081 and 022431102)
  6. FACS Tubes: 5 ml Polystyrene Round-bottom tube with 40 μm Cell Strainer tops (Corning, Falcon®, catalog number: 352235)
  7. 70 μm cell strainer (Fisher Scientific, catalog number: 22-363-548)
  8. 40 μm cell strainer caps
  9. Razor blades
  10. Plate
  11. Dry ice
  12. 2-Mercaptoethanol (BME) (Sigma-Aldrich, catalog number: M6250-100)
  13. RNasin Ribonuclease Inhibitors (Promega, catalog number: N2115)
  14. RNA Later (Life Technologies, catalog number: AM7020)
  15. Miltenyi Neural Dissociation Kit (NDK) (Papain) (MACS Miltenyi Biotec, Miltenyi, catalog number: 130-092-628)
    Note: Store at 2-8 °C, Shelf life: 24 months from the date of manufacture.
  16. Fc Block: Purified Rat Anti-Mouse CD16/CD32 (Fisher Scientific, BD Pharmingen, catalog number: BDB553142) (Store at 4 °C)
  17. CD31-APC: APC Rat Anti-Mouse CD31 (BD Biosciences, catalog number: 551262) (Store at 2-8 °C)
  18. CD45-APC/Cy7: APC/Cy7 Rat Anti-Mouse CD45 (BioLegend, catalog number: 103115) (Store at 2-8 °C)
  19. Or CD45-FITC: FITC Rat Anti-Mouse CD45 (BD Pharmingen, catalog number: 553080, Clone 30-F11)
  20. CD11b-BV421: Brilliant Violet 421 Rat Anti-Mouse CD11b (BioLegend, catalog number: 101235) (Store at 2-8 °C)
  21. Propidium Iodide (PI): Propidium Iodide Solution (Sigma-Aldrich, catalog number: P4864) (Store at 2-8 °C)
  22. RNeasy Plus Micro kit (QIAGEN, catalog number: 74034)
  23. Optional: Rat monoclonal anti-VCAM1 (Clone M/K-2.7, Bioxell, catalog number: BE0027)
  24. Optional: Rat IgG1 Isotype antibody (Clone HRPN, Bioxell, catalog number: BE0088)
  25. Optional: DyLightTM Antibody Labeling Kit (DyLightTM 488, Thermo Scientific, catalog number: 53025)
  26. Modified DPBS (mDPBS) (see Recipes)
    Note: Sterile filter solution, Store at 2-8 °C, Shelf life: 36 months from the date of manufacture.
    1. Dulbecco’s Phosphate-buffered Saline (DPBS) with Calcium and Magnesium (Thermo Fisher, GibcoTM, catalog number: 14040-133)
      Note: Store at 2-8 °C, Shelf life: 36 months from the date of manufacture.
    2. Glucose (1,000 mg/L) (Fisher Scientific, ACROS Organics, catalog number: 41095-5000)
    3. Sodium Pyruvate (30 mg/L) (Thermo Fisher, GibcoTM, catalog number: 11360-070)
  27. FACS buffer (see Recipes)
    Note: Sterile Filter solution, Store at 2-8 °C, Shelf life: 3 months from the date of manufacture.
    1. Modified DPBS
    2. 0.5% BSA (Fisher Scientific, catalog number: BP1600-100)
    3. 2 mM EDTA (Thermo Fisher, InvitrogenTM, catalog number: AM9912)
  28. 0.9 M Sucrose (see Recipes)
    Sucrose in DPBS (Fisher Scientific, catalog number: BP220-10)
    Note: Sterile Filter solution, Store at 2-8 °C, Shelf life: 3 months from the date of manufacture.

Equipment

  1. Graduated cylinder
  2. -80 °C freezer
  3. Shaker
  4. Centrifuge (Eppendorf, model: 5810R, catalog number: 022625004)
  5. Warm Water Bath (preheated at 37 °C) 
  6. Multi-Purpose Rotator (Thermo Fisher, Lab Rotator, catalog number: 2309-1CEQ)
  7. BD FACSAriaTM II or III cell sorter (BD Biosciences)
  8. Microscissors, fine forceps 
  9. Agilent 2100 Bioanalyzer (Agilent Technologies)

Software

  1. BD FACSDIVATM SOFTWARE (BD Biosciences, version: V8.0.1)
  2. FlowJoTM (© FlowJo, LLC, version: 9.9.4 or higher)

Procedure

Notes:

  1. Perform all centrifugation and staining steps at room temperature unless otherwise stated. Flow cytometry sorter should be set to 100 μm nozzle.
  2. Prepare all reagents ahead of time and warm to room temperature if necessary. 
  3. Optional: To label adhesion molecules, such as VCAM1 which is uniquely enriched in arterial and venous BECs (Vanlandewijck et al., 2018; Yousef et al., 2018), on BECs for vessel segmental enrichment. If VCAM1 (or another adhesion molecule) labeling is desired, conjugate anti-VCAM1 mAb and rat IgG1 isotype control antibody to DyLightTM-488 according to manufacturer’s instructions. 
  4. Mice: Mice were anesthetized with avertin and perfused with 20 ml cold PBS following blood collection. 
  5. Optional: Set controls using LPS-stimulated mice: Mice were treated with LPS for 16 h and 2 h before perfusion (1 mg/kg, i.p.). Mice were injected with fluorescently labeled anti-VCAM1 mAb (100 µg, r.o.) or IgG1 isotype control 2 h prior to cell isolation and flow cytometry analysis.
  6. All experimental (healthy) mice are also injected with anti-VCAM1 mAb (100 µg, r.o.) 2 h prior to cell isolation if vessel segmental enrichment is desired. 


  1. Tissue Dissociation
    1. For each sample, usually comprising of dissected cortex/hippocampus from 1-2 brains for applications such as bulk RNA-Seq, or pooled hippocampi (from 4 mice) for applications such as single cell RNA-Seq (Yousef et al., 2018), preheat 1.5 ml of Buffer X from Neural Dissociation Kit (NDK) + 9 μl of 2-Mercaptoethanol (BME) + 50 μl Enzyme P from NDK in 15 ml Falcon tubes in a 37 °C water bath.
    2. Remove meninges by rolling whole brain on Whatman paper.
    3. Dissect hippocampus, cortex, and remainder of the brain using microscissors and forceps in a sterile plate. Separate individual tissues and roughly chop into fine bits using a razor blade. Chop the brain segments to such a degree that you can pass the minced tissue through a trimmed, 1 ml pipettor (trimmed meaning the very tip is cut off to increase the diameter of the hole so that minced tissue can get through), but not so chopped that it becomes very mushy and could go through an untrimmed 1 ml pipettor. If the brain is too minced, it might be overly digested in the enzyme mixture in subsequent steps leading to cell loss. 
    4. Alternatively, if isolating the hippocampus or other very small brain regions separately, it is not necessary to mince in a sterile plate. Rather, transfer each dissected small tissue using sterile forceps into 1.5 ml Eppendorf tubes with 0.5 ml of Buffer X from NDK and chop finely with microscissors.
    5. Using a trimmed 1 ml tip (trimmed meaning the very tip is cut off to slightly increase the diameter of the hole), transfer finely chopped samples to the preheated Buffer X solution prepared in Step A1. Triturate 10 times with the same pipette tip. Incubate in a 37 °C water bath for 15 min. Flick tubes every 5 min of incubation. Prepare Enzyme 2 Mix from NDK for each sample: 10 μl of Enzyme A + 20 μl Buffer Y.
    6. Add 30 μl of Enzyme 2 Mix into each sample and triturate 10 times with a 1 ml pipette tip, start 10 min timer from when the first sample receives the Enzyme 2 Mix. Incubate samples in a 37 °C water bath for the remainder of the 10 min timer, flick samples every 5 min.
      Note: Be quick! Do not over incubate, over digestion will result in poor yields.
    7. Prepare 50 ml tubes with 70 μm cell strainers, wash strainers with 1 ml of mDPBS. Collect the flowthrough in the 50 ml tube. 
    8. Triturate samples 10 times with a 1 ml pipette tip and immediately add 10 ml mDPBS.
    9. Pass all of the supernatant through a 70 μm cell strainer prepared in Step A7. Wash through another 5 ml mDPBS.
    10. Centrifuge at 300 x g for 10 min. Discard the supernatant and collect the pellet. Carefully pipette out supernatant without disturbing the pellet.

  2. Myelin Removal and Staining
    1. Resuspend pellets in 5 ml (2.5 ml for Hippocampus samples) of 0.9 M Sucrose and transfer each pellet to a 15 ml tube. Centrifuge at 850 x g for 15 min. Discard the supernatant, and collect the pellet. Carefully pipette out and discard the supernatant without disturbing the pellet.
    2. Sample resuspension
      1. For Hippocampus samples:
        Resuspend pellet in 2 ml FACS buffer, transfer to a 2 ml Eppendorf tube and wait for other tissue type samples before continuing to Step B3.
      2. For Cortex and Whole Brain samples:
        Resuspend pellet in 1 ml 0.9 M Sucrose before further adding another 2 ml of Sucrose. Centrifuge at 850 x g for 15 min. Discard supernatant without disturbing the pellet. Resuspend the pellet in 2 ml FACS buffer, transfer to a 2 ml Eppendorf tube.
    3. Centrifuge all samples at 300 x g for 5 min. Prepare Fc Blocking solution (100 µl per sample):
      For 10 samples, dilute 10 μl Fc Block in 1 ml FACS buffer. Plan which Compensation standards will be needed (Single Channel comps for each marker, and 2 unstained controls, with and without propidium iodide (PI) which is added in Step C3 below). Cell pellets are visible for cortex pooled samples, but likely not visible for hippocampal samples. Note the estimated cell numbers in Step C6 below. 
    4. Resuspend each sample in 100 μl of Fc Blocking solution prepared in Step B3 above (For the sample to be used in Compensation standards, add an extra 25 μl of Fc Blocking solution to the sample for each planned Compensation standard). Incubate on a shaker for 5-10 min at room temperature.
    5. Add the following antibodies (1:100) and incubate on a shaker for 30 min at RT:
      Anti-CD31-APC
      Anti-CD11b-BV421
      Anti-CD45-APC/Cy7 or anti-CD45-FITC
      Note: If mice were injected with fluorescently labeled anti-mouse VCAM1-DyLightTM488 as described above, stain CD45 in the APC/Cy7 channel, and also gate CD31+VCAM1+ cells in the APC and FITC channels. 
    6. Add 1.8 ml of FACS buffer to dilute the samples, then centrifuge at 300 x g for 5 min.
    7. Prepare Sorting Mix: FACS buffer + PI (1:5,000 of a 1 mg/ml stock) + RNase Inhibitor (1:500) (For 5 ml: Add 1 μl PI and 10 μl RNase Inhibitor). 
    8. Carefully pipette out supernatant and resuspend each sample (except Compensation standards, resuspend comps in 0.5 ml FACS buffer) in 0.5 ml of Sorting Mix.
    9. Transfer all samples through cell strainer capped FACS tubes (Blue 40 μm cell strainer caps). Keep on ice and covered from light until sorted by flow cytometry. 
    10. If collecting whole population cells: Prepare 1.5 ml RNase-free collection tubes with 0.5 ml of RNA later. Be prepared to dilute samples to a total volume of 1.3 ml with RNA later after collecting cells and snap freeze samples with dry ice. 
    11. If collecting single cells: have plates prepared with RNA lysis buffer as instructed by manufacturer for the technique chosen, and kept frozen and stored at -80 °C until used for cell sorting (aka, Smart-seq-2 protocol as described previously [Picelli et al., 2014; Darmanis et al., 2015], was used in Yousef et al. [2018] study). 
    Notes: 
    1. This protocol does not cover detailed instructions for FACS parameter setup. For assistance, please contact your FACS core facility manager or FACS machine vendor.
    2. Use a 100 µm nozzle.
    3. While sorting, keep the threshold rate around or below 100x the value of the set frequency (typically fewer than 2,800 events per second).
    4. Estimated sort time is 15-20 min per stained sample.

  3. FACS and sample collection
    1. Run unstained cells to set up forward (FSC-A) and side scatter (SSC-A) (Figure 2B).
    2. Run single-color controls to set FACS parameters and compensate for channel spillover.
    3. Add 1:5,000 PI from a stock of 1 mg/ml (0.2 µg/ml final concentration) (or 1:500 from 1:10 pre-dilution) PI for live/dead staining right before running the individual sample, if it was not already added at Step B7 of Myelin Removal and Staining section above. 
    4. For your first run and whenever obtaining a new antibody: Run controls in which cells are stained with all antibodies except for one, to determine positive cell populations and set up gates accordingly. 
    5. Run stained FACS sample:
      1. Gate cells on forward (FSC-A = size) and sideward scatter (SSC-A = internal structure) to exclude cell debris and residual myelin
      2. Plot FSC-A against FSC-W to discriminate single cells from cell doublets/aggregates.
      3. Exclude PI positive (dead) cells.
      4. Exclude CD11b+CD45+ monocytes/macrophages and microglial cells by gating on CD45 and CD11b negative cells.
      5. Gate on CD31+ BECs and collect cells from this gate to obtain a pure brain endothelial cell population. Record at least 1 x 106 events.
    6. Collect cells in either: 
      1. 1.5 ml RNase-free tubes containing FACS buffer (if RNA integrity is less of a concern) or 0.5 ml RNA later (to preserve for later RNA isolation for bulk RNA-Seq). If containing 0.5 ml of RNA later: 
        1. Ideally, 100,000 cells or more are needed for optimal RNA extraction. Top off the RNA later up to 1.3 ml before mixing well and snap freezing the samples with dry ice. Store at -80 °C until RNA isolation. 
        2. When spinning down the cells to extract RNA, thaw cells and warm to room temperature before 10 min centrifugation at 1,000 x g. Isolate total RNA from the cell pellets using the RNeasy Plus Micro kit (QIAGEN). Assess RNA quantities and RNA quality using the Agilent 2100 Bioanalyzer (Agilent Technologies). All samples passed a quality control threshold (RIN ≥ 8.5) to proceed to library preparations and RNA-Seq.
      2. Single cells in a 96-well plate with RNA lysis buffer for single cell RNA-Seq (according to Smart-Seq-2 [Picelli et al., 2014; Darmanis et al., 2015] or other preferred protocol).
        Notes:
        1. Keep plates frozen on dry ice until just before they are used to sort cells. Immediately after cell sorting, cover each plate with an aluminum freezing compatible lid, vortex the plate to mix cells with RNA lysis buffer, and freeze plate on dry ice until it can be transferred and stored at -80 °C until RNA isolation. 
        2. For a typical sample of 1-2 cortex/hippocampi to isolate cells in bulk for RNA extraction (aka for bulk RNA-Seq), sorting by flow cytometry will result in around 100,000 isolated CD31+BECs per sample. For a typical sample of 4 pooled hippocampi, sorting by flow cytometry results in collection of a few thousand to tens of thousands of cells which varies by prep, cell collection method (bulk or single cell) and if VCAM1 is enriched. 

Data analysis

  1. Please refer to Yousef et al. (2018) published in BioRxiv for the data analysis
    This BEC isolation protocol was used in the following figures in the Yousef et al. (2018) paper:
    Figure 1a-c: Bulk RNA-Seq of young and aged cortex/hippocampal BECs.
    Supplemental Figure 1a-e: Bulk RNA-Seq of young and aged cortex/hippocampal BECs.
    Supplemental Figure 1f-k: C57BL6 mice were injected with anti-VCAM1-DL488 or IgG-DL488 isotype control (r.o.) 2 h before perfusion to label BECs in vivo prior to brain dissociation, staining, and FACS. The hippocampal cells were then used for single cell RNA-Seq (Figure 2 and Supplemental Figure 2).
    Figure 2 and Supplemental Figure 2: Single cell RNA-Seq of BECs isolated from the hippocampus.
    Figure 3c-d: BECs isolated from young mice treated with young or aged plasma and injected with anti-VCAM1-DL488 or IgG-DL488 isotype control (r.o.) 2 h before perfusion to label BECs in vivo prior to brain dissociation, staining, and FACS. Young mice stimulated with LPS were used to set the gates. 
  2. Please refer to “Collagenase-based Single Cell Isolation of Primary Murine Brain Endothelial Cells Using Flow Cytometry” by Czupalla et al. (2018) Bio-Protocol, for an alternative protocol for BEC cell isolation by flow cytometry which uses a more gentle collagenase-based method and several markers to stain additional cell types. This protocol was used for Supplemental Figure 7a-d in the Yousef et al. (2018) paper published in BioRxiv to show genetic recombination and deletion of Vcam1 in tamoxifen-treated Vcam1fl/flSlco1c1-CreERT2+/- mice.

Recipes

  1. Modified DPBS (mDPBS) (500 ml) 
    1. Measure 500 mg of glucose in a graduated cylinder
    2. Add 1.36 ml of 100 mM sodium pyruvate to the graduated cylinder, and then add DPBS up to the 500 ml notch
    3. Seal the graduated cylinder using parafilm and mix thoroughly by inverting several times
    4. Once mixed, sterile filter using a Thermo Scientific Nalgene Rapid-Flow Sterile filter unit with 0.2 μm pore
  2. FACS buffer (500 ml)
    1. Add 2.5 g of BSA and 2 ml of 0.5 M EDTA to 500 ml of mDPBS
    2. Mix thoroughly and sterile filter using a Thermo Scientific Nalgene Rapid-Flow Sterile filter unit with 0.2 μm pore
  3. 0.9 M Sucrose (500 ml)
    1. Measure 154.04 g of sucrose in a graduated cylinder
    2. Add DPBS up to the 500 ml notch
    3. Seal the graduated cylinder using parafilm and mix thoroughly by inverting several times
    Optional: If the mixture is not completely in solution, lightly warm up the solution in a heated water bath and mix. Once mixed, sterile filter using a Thermo Scientific Nalgene Rapid-Flow Sterile filter unit with 0.2 μm pore.

Acknowledgments

We thank Lusijah Sutherland PhD, and Corey Cain PhD, for managing the core flow cytometry facility at the VA in Palo Alto and providing H.Y. and C.J.C. training on the instruments; Corey Cain PhD as well for his experimental advice, assistance with flow cytometry and analysis of PBMCs and thoughtful discussion. We would also like to thank Ryan Watts PhD and Nga Bien-Ly PhD for sharing the original BEC isolation protocol used for RNA-Seq. This work was funded by the Department of Veterans Affairs (T.W.-C.), the National Institute on Aging (SPO: 116650; 1F32AG051330-01A1 to H.Y., R01-AG045034 and DP1-AG053015 to T.W.-C.), the NOMIS Foundation (T.W.-C.), The Glenn Foundation for Aging Research (T.W.-C), a SPARK grant to H.Y. through the Stanford Clinical and Translational Science Award (CTSA) to Spectrum (UL1 TR001085), the National Institutes of Health (R01-GM37734 and R37-AI047822 to E.C.B, RO1 AI109452 to HH), the Stanford Institute for Immunity, Transplantation and Infection (C.J.C.), and the Edinger Institute (C.J.C.). The CTSA program is led by the National Center for Advancing Translational Sciences (NCATS) at the National Institutes of Health (NIH).

Competing interests

There are no competing conflicts of interest.

Ethics

The mice used in this protocol were bred and aged in-house and lived under a 12 h light-dark cycle in pathogenic-free conditions, in accordance with the Guide for Care and Use of Laboratory Animals of the National Institutes of Health.

References

  1. Abbott, N. J., Patabendige, A. A., Dolman, D. E., Yusof, S. R. and Begley, D. J. (2010). Structure and function of the blood-brain barrier. Neurobiol Dis 37(1): 13-25.
  2. Banks, W. A. (2016). From blood-brain barrier to blood-brain interface: new opportunities for CNS drug delivery. Nat Rev Drug Discov 15(4): 275-292.
  3. Czupalla, C. J., Yousef, H., Wyss-Coray, T. and Butcher, E. C. (2018). Collagenase-based single cell isolation of primary murine brain endothelial cells using flow cytometry. Bio-protocol 8(22): e3092.
  4. Daneman, R., Zhou, L., Agalliu, D., Cahoy, J. D., Kaushal, A. and Barres, B. A. (2010). The mouse blood-brain barrier transcriptome: a new resource for understanding the development and function of brain endothelial cells. PLoS One 5(10): e13741.
  5. Darmanis, S., Sloan, S. A., Zhang, Y., Enge, M., Caneda, C., Shuer, L. M., Hayden Gephart, M. G., Barres, B. A. and Quake, S. R. (2015). A survey of human brain transcriptome diversity at the single cell level. Proc Natl Acad Sci U S A 112(23): 7285-7290.
  6. Engelhardt, B., Vajkoczy, P. and Weller, R. O. (2017). The movers and shapers in immune privilege of the CNS. Nat Immunol 18(2): 123-131.
  7. Picelli, S., Faridani, O. R., Bjorklund, A. K., Winberg, G., Sagasser, S. and Sandberg, R. (2014). Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9(1): 171-181.
  8. Tam, S. J., Richmond, D. L., Kaminker, J. S., Modrusan, Z., Martin-McNulty, B., Cao, T. C., Weimer, R. M., Carano, R. A., van Bruggen, N. and Watts, R. J. (2012). Death receptors DR6 and TROY regulate brain vascular development. Dev Cell 22(2): 403-417.
  9. Vanlandewijck, M., He, L., Mae, M. A., Andrae, J., Ando, K., Del Gaudio, F., Nahar, K., Lebouvier, T., Lavina, B., Gouveia, L., Sun, Y., Raschperger, E., Rasanen, M., Zarb, Y., Mochizuki, N., Keller, A., Lendahl, U. and Betsholtz, C. (2018). A molecular atlas of cell types and zonation in the brain vasculature. Nature 554(7693): 475-480.
  10. Yousef, H., Czupalla, C. J., Lee, D., Burke, A., Chen, M., Zandstra, J., Berber, E., Lehallier, B., Mathur, V., Nair, R. V., Bonanno, L., Merkel, T., Schwaninger, M., Quake, S., Butcher, E. C. and Wyss-Coray, T. (2018). Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier. bioRxiv. doi:10.1101/242198.

简介

脑内皮细胞(BEC)形成血脑屏障(BBB)的组成部分,其将全身环境与脑实质分开并保护大脑免受病原体和循环因子的影响。为了研究BEC生物学,特别感兴趣的是建立一种方法,使研究人员能够研究和理解在体内平衡,衰老和疾病期间调节其功能的潜在分子机制。此外,由于脑血管系统的异质性和构成BBB的不同血管类型,特别感兴趣的是从脑的各个亚区域分离用于单细胞分析的原代BEC,例如神经源性和高度血管化的海马体并且富集特定船只类型。过去,分离内皮细胞的方法依赖于转基因小鼠,并且通常导致纯度不足的细胞群和低产量。该协议描述了一种允许使用荧光激活细胞分选(FACS)单细胞分离高纯度脑内皮细胞群的技术。简而言之,在灌注和小心移除脑膜并解剖皮质/海马后,将脑组织机械均质化并酶促消化,得到单细胞悬浮液。细胞用荧光染料缀合的抗体染色,鉴定CD31 +脑内皮细胞,以及CD45 + CD11b +髓样细胞用于排除。使用流式细胞术分离细胞群,然后将CD31 + BEC大量分选成RNA或作为单个细胞直接分选到RNA裂解缓冲液中用于单一或大量RNA-Seq分析。该方案不需要表达转基因来标记脑内皮细胞,因此可以应用于任何小鼠模型。在我们的手中,该方案具有高度可重复性,平均产量为从成年小鼠皮质/海马体分离的1×10 5个细胞。

【背景】 血脑屏障(BBB)是一种复杂的血管结构,可以分离和保护大脑免受全身循环因子和免疫细胞的影响,同时允许选择性运输关键营养素。这种高度专业化的脉管系统调节代谢物穿过屏障的选择性运输。 BEC还形成神经血管单元(NVU)的中心组件,与大脑中的各种细胞类型密切相关和串扰,包括神经前体细胞,小胶质细胞和脑驻留免疫细胞。 BBB不仅限制离子和其他分子(如葡萄糖)的通过,还可以防止血液和脑实质之间毒素,细菌,病毒和细胞的不受控制的交换(Abbott et al。,2010 )。富含营养素的含氧血液通过脑动脉BEC(动脉和小动脉)泵入大脑,这些BEC受到平滑肌细胞(SMC)的保护和支持,平滑肌细胞覆盖内皮并形成由天体细胞末端分层的基底膜。脑实质(图1)。将血液转移到高度特化的毛细血管中,毛细血管由形成独特紧密连接的BEC组成,并被内皮基底膜内的周细胞(Peric。)包裹,然后由星形胶质细胞末端足部覆盖。 BBB毛细血管是控制流体和溶质进入CNS的部位。白细胞(Leuk。)进入CNS实质的免疫监视和偶然外渗发生在血管段后毛细血管静脉细胞(小静脉和静脉)的水平,血液在通过毛细血管后流入血管段。毛细血管后小静脉在内皮细胞和星形胶质细胞基底膜之间含有增大的血管周围空间,偶尔存在免疫细胞(Banks,2016; Engelhardt et al。,2017)。

概述的荧光激活细胞分选(FACS)单细胞分离方法基于先前描述的BEC分离程序(Tam 等人,,2012),其已经在研究的背景下进一步开发和修改。旨在确定老化和暴露于老年血液时脑内皮细胞中发生的分子变化(Yousef et al。,2018)。图2显示了BEC分离的FACS程序和细胞门控实例的示意图。衰老导致涉及内皮细胞活化的慢性炎症过程的上调,这有助于降低神经前体细胞活性并增加衰老大脑中的神经炎症( Yousef et al。,2018)。该技术成功应用于研究老年系统性环境对海马神经发生和小胶质细胞激活的影响(Yousef et al。,2018)。该研究强调了血管细胞粘附分子1(VCAM1)作为成人神经发生和小胶质细胞活性诱导物的负调节因子的作用。 VCAM1仅在基线条件下在非常小的细胞亚群中表达,因此,使用常规的两步抗体染色未充分免疫标记以进行FACS分析。为此,用来自鼠伤寒沙门氏菌的脂多糖(LPS)全身刺激小鼠以增加VCAM1表达。然后在小鼠眼内注射荧光标记的抗小鼠VCAM1,这导致可靠地检测VCAM1阳性脑内皮细胞亚群。 LPS刺激小鼠中的CD31 + VCAM1 + BEC可用于在流式分选仪中设置正门和负门以定量评估CD31 + VCAM1 +在正常年轻和年老小鼠,或年轻或老年血浆处理的幼鼠中的表达,并分离这种罕见的亚群(Yousef et al。,2018;图3) 。此外,VCAM1富含动脉和静脉BEC,可用于富集和研究这两个血管节段性群体(Vanlandewijck et al。,2018; Yousef et al。, 2018)。

在过去的十年中,已经采用了几种用于脑内皮细胞分离的方案,这些方案倾向于使用用荧光蛋白如GFP标记的转基因内皮细胞(Daneman et al。,2010; Vanlandewijck et al。,2018)。因为这些技术依赖于转基因内皮细胞标记物,所以它们可能不容易应用于患病或正常老化的小鼠模型。该方案允许使用商业神经解离试剂盒(MACS Miltenyi Biotec,Miltenyi)使用基于木瓜蛋白酶的酶消化从任何小鼠品系或鼠动物模型中分离高纯度脑内皮细胞群。
图1.血脑屏障示意图富含营养素的含氧血液通过脑动脉BEC(动脉和小动脉)泵入大脑,这些BEC由平滑肌细胞保护和支持(SMC) )覆盖内皮并形成由脑实质的星形胶质细胞末端分层的基底膜。将血液转移到高度特化的毛细血管中,毛细血管由形成独特紧密连接的BEC组成,并被内皮基底膜内的周细胞(Peric。)包裹,然后由星形胶质细胞末端足部覆盖。 BBB毛细血管是控制流体和溶质进入CNS的部位。白细胞(Leuk。)进入CNS实质的免疫监视和偶然外渗发生在血管段后毛细血管静脉细胞(小静脉和静脉)的水平,血液在通过毛细血管后流入血管段。毛细血管后小静脉在内皮细胞和星形胶质细胞基底膜之间含有增大的血管周围空间,偶尔存在免疫细胞(Banks,2016; Engelhardt et al。,2017)。 (采用Yousef 等人 [2018]图2a)


图2.通过流式细胞术分离小鼠脑内皮细胞。 A.来自小鼠皮层和海马的CD31 + CD45 - BECs的流式分选示意图。每个分离的RNA样品是来自2个小鼠脑的BEC库。 B. FACS门控分离单个BEC的策略。排除PI +死细胞。门控CD11b +和CD45 +细胞以排除单核细胞/巨噬细胞和小胶质细胞。 CD31 + Cd11b - CD45 -细胞被定义为BEC群体。 (采用Yousef 等人 [2018]补充图1a-b)


图3.通过流式细胞术鉴定VCAM1 + CD31 + BECs。 Vcam1 fl / fl < / em> Slco1c1-Cre ERT2 +/-小鼠(Cre +)每天一次接受他莫昔芬(100mg / kg; ip),持续5天。休息3天后,在细胞分离前,用LPS处理小鼠16小时和灌注前2小时(1mg / kg,ip)和荧光标记的抗VCAM1 mAb(100μg,ro)2小时。流式细胞术分析。从门控图分离的CD31 + VCAM1 +细胞的门控图是他莫昔芬处理的,LPS刺激的老年(19个月大)Slco1c1-Cre ERT2 + / - -Vcam1 FL / FL(酶Cre +)和缺乏酶Cre基因(Cre-)中,用荧光标记DL488抗VCAM1单抗或IgG-DL488注入的副本同窝对照小鼠在处死前2小时进行同种型对照(ro)。

关键字:脑内皮细胞, 血脑屏障, 神经血管单元, 单细胞分离, 细胞分选

材料和试剂

  1. 移液器吸头必须无菌且保留率低
  2. Whatman论文:GE Healthcare Whatman定量滤纸40级(Fisher Scientific,GE Healthcare,目录号:09-927-541)
  3. 带有PES膜的Thermo Scientific Nalgene快速流动无菌一次性过滤装置(Thermo Fisher,目录号:569-0020)
  4. 15毫升和50毫升管(康宁,目录号:430790和430828)
  5. 1.5毫升和2.0毫升Eppendorf管蛋白低结合(Eppendorf,目录号:022431081和022431102)
  6. FACS管:5 ml聚苯乙烯圆底管,40μm细胞过滤器顶部(Corning,Falcon®,目录号:352235)
  7. 70μm细胞过滤器(Fisher Scientific,目录号:22-363-548)
  8. 40μm细胞过滤器盖
  9. 剃刀片
  10. 盘子
  11. 干冰
  12. 2-巯基乙醇(BME)(西格玛奥德里奇,目录号:M6250-100)
  13. RNasin Ribonuclease Inhibitors(Promega,目录号:N2115)
  14. RNA Later(Life Technologies,目录号:AM7020)
  15. Miltenyi神经解离试剂盒(NDK)(木瓜蛋白酶)(MACS Miltenyi Biotec,Miltenyi,目录号:130-092-628)
    注意:储存在2-8°C,保质期:自生产之日起24个月。
  16. Fc块:纯化的大鼠抗小鼠CD16 / CD32(Fisher Scientific,BD Pharmingen,目录号:BDB553142)(在4°C储存)
  17. CD31-APC:APC大鼠抗小鼠CD31(BD Biosciences,目录号:551262)(在2-8°C保存)
  18. CD45-APC / Cy7:APC / Cy7大鼠抗小鼠CD45(BioLegend,目录号:103115)(2-8°C储存)
  19. 或CD45-FITC:FITC大鼠抗小鼠CD45(BD Pharmingen,目录号:553080,克隆30-F11)
  20. CD11b-BV421:Brilliant Violet 421大鼠抗小鼠CD11b(BioLegend,目录号:101235)(2-8°C储存)
  21. 碘化丙啶(PI):碘化丙啶溶液(Sigma-Aldrich,目录号:P4864)(2-8°C保存)
  22. RNeasy Plus Micro试剂盒(QIAGEN,目录号:74034)
  23. 可选:大鼠单克隆抗VCAM1(克隆M / K-2.7,Bioxell,目录号:BE0027)
  24. 可选:大鼠IgG1同种型抗体(克隆HRPN,Bioxell,目录号:BE0088)
  25. 可选: DyLight TM抗体标记试剂盒(DyLight TM 488,Thermo Scientific,目录号:53025)
  26. 改良DPBS(mDPBS)(见食谱)
    注意:无菌过滤器溶液,储存在2-8°C,保质期:自生产之日起36个月。
    1. Dulbecco的磷酸盐缓冲盐水(DPBS)与钙和镁(Thermo Fisher,Gibco TM,目录号:14040-133)
      注意:储存在2-8°C,保质期:自生产之日起36个月。
    2. 葡萄糖(1,000 mg / L)(Fisher Scientific,ACROS Organics,目录号:41095-5000)
    3. 丙酮酸钠(30 mg / L)(Thermo Fisher,Gibco TM,目录号:11360-070)
  27. FACS缓冲液(见食谱)
    注意:无菌过滤器溶液,储存在2-8°C,保质期:自生产之日起3个月。
    1. 修改后的DPBS
    2. 0.5%BSA(Fisher Scientific,目录号:BP1600-100)
    3. 2 mM EDTA(Thermo Fisher,Invitrogen TM,目录号:AM9912)
  28. 0.9 M蔗糖(见食谱)
    蔗糖在DPBS(Fisher Scientific,目录号:BP220-10)
    注意:无菌过滤器溶液,储存在2-8°C,保质期:自生产之日起3个月。

设备

  1. 刻度量筒
  2. -80°C冰箱
  3. 振动筛
  4. 离心机(Eppendorf,型号:5810R,目录号:022625004)
  5. 温水浴(预热37°C)&nbsp;
  6. 多功能旋转器(Thermo Fisher,Lab Rotator,目录号:2309-1CEQ)
  7. BD FACSAria TM II或III细胞分选仪(BD Biosciences)
  8. 微型剪刀,精细钳子&nbsp;
  9. Agilent 2100生物分析仪(安捷伦科技)

软件

  1. BD FACSDIVA TM软件(BD Biosciences,版本:V8.0.1)
  2. FlowJo TM(©FlowJo,LLC,版本:9.9.4或更高版本)

程序

注意:

  1. 除非另有说明,否则在室温下进行所有离心和染色步骤。流式细胞分选仪应设置为100μm喷嘴。
  2. 提前准备所有试剂并在必要时温热至室温。
  3. 可选:标记粘附分子,如VCAM1,其独特地富集动脉和静脉BEC(Vanlandewijck 等 ,2018; Yousef et al。 ,2018),关于BEC血管节段富集。如果需要VCAM1(或其他粘附分子)标记,缀合抗VCAM1 mAb和大鼠IgG1同种型对照抗体DyLight TM -488根据制造商的说明。&nbsp;
  4. 小鼠:小鼠用阿佛丁麻醉后,在采血后用20毫升冷PBS灌注。
  5. 可选: 使用LPS刺激的小鼠设置对照:小鼠在灌注前用LPS处理16小时和2小时(1mg / kg,腹膜内)。在细胞分离和流式细胞术分析之前2小时,给小鼠注射荧光标记的抗VCAM1 mAb(100μg,r.o。)或IgG1同种型对照。
  6. 如果需要进行血管节段富集,所有实验性(健康)小鼠在细胞分离前2小时也注射抗VCAM1 mAb(100μg,r.o。)。


  1. 组织解离
    1. 对于每个样品,通常包括来自1-2个脑的解剖的皮层/海马体,用于诸如大量RNA-Seq或汇集的海马(来自4只小鼠)的应用,用于诸如单细胞RNA-Seq的应用(Yousef 等。 ,2018),在神经离解试剂盒(NDK)中预热1.5 ml缓冲液X +在15°F的水浴中,在15 ml Falcon试管中从NDK中加入9μl2-巯基乙醇(BME)+50μl酶P 。
    2. 在Whatman纸上滚动全脑去除脑膜。
    3. 使用微型剪刀和镊子在无菌板中解剖海马,皮质和大脑的其余部分。使用剃刀刀片分离单个组织并大致切成细小的碎片。将大脑节段切割到这样的程度,即你可以将切碎的组织穿过修剪过的1毫升移液器(修剪后意味着切割尖端以增加孔的直径以使切碎的组织能够通过),但不是这样切碎,它变得非常糊状,可以通过一个未修剪的1毫升移液器。如果大脑过于剁碎,可能会在后续步骤中过度消化酶混合物,导致细胞丢失。&nbsp;
    4. 或者,如果分开分离海马或其他非常小的脑区域,则不必在无菌平板中切碎。相反,使用无菌镊子将每个切开的小组织转移到1.5ml含有0.5ml来自NDK的缓冲液X的Eppendorf管中,并用微型剪刀精细切碎。
    5. 使用修剪的1毫升尖端(修剪意味着切割尖端以略微增加孔的直径),将精细切碎的样品转移到步骤A1中制备的预热缓冲液X溶液中。用相同的移液管尖端研磨10次。在37°C水浴中孵育15分钟。每5分钟孵育一次。从NDK为每个样品制备酶2混合物:10μl酶A +20μl缓冲液Y.
    6. 在每个样品中加入30μl酶2混合液,用1 ml移液管尖端研磨10次,从第一个样品接受酶2混合物开始10分钟计时。将样品在37°C水浴中孵育10分钟计时器的剩余时间,每5分钟轻弹一次样品。
      注意:快点!不要过度孵化,过度消化会导致产量低下。
    7. 准备50毫升管与70微米细胞过滤器,洗涤过滤器与1毫升mDPBS。将流通物收集在50毫升管中。&nbsp;
    8. 用1ml移液管尖端研磨样品10次,并立即加入10ml mDPBS。
    9. 将所有上清液通过步骤A7中制备的70μm细胞过滤器。用另外5ml mDPBS洗涤。
    10. 在300 x g 下离心10分钟。 丢弃上清液并收集沉淀。小心吸取上清液,不要打扰沉淀。

  2. 髓鞘去除和染色
    1. 将颗粒重悬于5ml(海马样品2.5ml)0.9M蔗糖中,并将每个沉淀转移至15ml管中。在850 x g 下离心15分钟。 丢弃上清液,收集沉淀物。小心吸取并丢弃上清液,不要打扰沉淀。
    2. 样品重新悬浮
      1. 对于海马样本:
        将沉淀重悬于2ml FACS缓冲液中,转移至2ml Eppendorf管中并等待其他组织类型样品,然后继续进行步骤B3。
      2. 对于Cortex和Whole Brain样本:
        将沉淀重悬于1ml 0.9M蔗糖中,然后再加入另外2ml蔗糖。在850 x g 下离心15分钟。丢弃上清液,不要打扰沉淀。将沉淀重悬于2ml FACS缓冲液中,转移至2ml Eppendorf管中。
    3. 将所有样品在300 x g 下离心5分钟。准备Fc阻断溶液(每个样品100μl):
      对于10个样品,在1ml FACS缓冲液中稀释10μlFcBlock。计划需要哪些补偿标准(每个标记的单通道补偿,以及2个未染色的对照,有和没有碘化丙啶(PI),在下面的步骤C3中添加)。对于皮质合并的样品,细胞沉淀是可见的,但对于海马样品可能不可见。请注意下面步骤C6中的估计单元格数。&nbsp;
    4. 将每个样品重悬于上述步骤B3中制备的100μlFc阻断溶液中(对于用于补偿标准品的样品,对于每个计划的补偿标准,向样品中添加额外的25μlFc阻断溶液)。在室温下在振荡器上孵育5-10分钟。
    5. 加入以下抗体(1:100)并在摇床上孵育30分钟:
      抗CD31-APC
      抗CD11b-BV421
      抗CD45-APC / Cy7或抗CD45-FITC
      注意:如果小鼠如上所述注射荧光标记的抗小鼠VCAM1-DyLight TM 488,则在APC中染色CD45 / Cy7通道,以及APC和FITC通道中的门CD31 + VCAM1 +细胞。&nbsp;
    6. 加入1.8ml FACS缓冲液稀释样品,然后以300 x g 离心5分钟。
    7. 准备分选混合物:FACS缓冲液+ PI(1:5,000的1 mg / ml原液)+ RNase抑制剂(1:500)(对于5 ml:加入1μlPI和10μlRNase抑制剂)。&nbsp;
    8. 小心吸取上清液,将每个样品(补偿标准除外,重悬于0.5 ml FACS缓冲液中)重悬于0.5 ml分选混合液中。
    9. 通过细胞过滤器加盖的FACS管(蓝色40μm细胞过滤器盖)转移所有样品。保持在冰上并避光,直至通过流式细胞仪分选。&nbsp;
    10. 如果收集整个群体细胞:随后准备1.5毫升无RNA酶的收集管和0.5毫升RNA。准备好在收集细胞后用RNA稀释样品至总体积为1.3 ml,并用干冰快速冷冻样品。&nbsp;
    11. 如果收集单个细胞:按照制造商的指示用所选技术制备用RNA裂解缓冲液制备的平板,并保持冷冻并储存在-80℃直至用于细胞分选(aka,如前所述的Smart-seq-2方案[Picelli] et al。,2014; Darmanis et al。,2015],用于Yousef et al。 [2018]研究)。&nbsp;
    注意:&nbsp;
    1. 此协议未涵盖FACS参数设置的详细说明。如需帮助,请联系您的FACS核心设施经理或FACS机器供应商。
    2. 使用100μm喷嘴。
    3. 在排序时,将阈值速率保持在设定频率值的100倍左右(通常每秒少于2,800个事件)。
    4. 每个染色样品的估计分拣时间为15-20分钟。

  3. FACS和样品采集
    1. 运行未染色的细胞以设置前向(FSC-A)和侧向散射(SSC-A)(图2B)。
    2. 运行单色控件以设置FACS参数并补偿通道溢出。
    3. 在运行单个样品之前,从1 mg / ml(0.2μg/ ml终浓度)(或1:500 1:10预稀释)PI中加入1:5,000 PI进行活/死染色,如果是尚未在上面的髓磷脂去除和染色部分的步骤B7中添加。&nbsp;
    4. 对于您的第一次运行以及每当获得新抗体时:运行对照,其中细胞用除1之外的所有抗体染色,以确定阳性细胞群并相应地设置门。&nbsp;
    5. 运行染色的FACS样品:
      1. 门前细胞(FSC-A =大小)和侧向散射(SSC-A =内部结构)以排除细胞碎片和残留髓鞘
      2. 绘制针对FSC-W的FSC-A以区分单细胞和细胞双联体/聚集体。
      3. 排除PI阳性(死亡)细胞。
      4. 通过门控CD45和CD11b阴性细胞排除CD11b + CD45 +单核细胞/巨噬细胞和小神经胶质细胞。
      5. 在CD31 + BEC上接门并从该门收集细胞以获得纯脑内皮细胞群。记录至少1 x 10 6事件。
    6. 收集细胞:&nbsp;
      1. 1.5毫升不含RNase的试管含有FACS缓冲液(如果RNA完整性不太重要)或0.5毫升RNA后(保留以后用于大量RNA-Seq的RNA分离)。如果以后含有0.5毫升RNA:&nbsp;
        1. 理想情况下,最佳RNA提取需要100,000个细胞或更多细胞。然后将RNA加完至1.3毫升,然后充分混合并用干冰快速冷冻样品。储存在-80°C直至RNA分离。&nbsp;
        2. 当旋转细胞以提取RNA时,解冻细胞并温热至室温,然后在1,000 x g 离心10分钟。使用RNeasy Plus Micro试剂盒(QIAGEN)从细胞沉淀中分离总RNA。使用Agilent 2100生物分析仪(Agilent Technologies)评估RNA量和RNA质量。所有样品均通过质量控制阈值(RIN≥8.5)以进行文库制备和RNA-Seq。
      2. 96孔板中的单细胞,RNA裂解缓冲液用于单细胞RNA-Seq(根据Smart-Seq-2 [Picelli et al。,2014; Darmanis et al。,2015]或其他首选协议)。
        注意:
        1. 将板冷冻在干冰上,直到它们用于分选细胞。在细胞分选后立即用铝冷冻相容的盖子盖住每个平板,涡旋平板以将细胞与RNA裂解缓冲液混合,并在干冰上冷冻平板直至其可以转移并储存在-80℃直至RNA分离。&nbsp;
        2. 对于典型的1-2皮层/海马体样本来分离大量细胞用于RNA提取(也称为大量RNA-Seq),通过流式细胞术分选将导致大约100,000个分离的CD31 + 每个样本的BEC。对于4个汇集的海马的典型样本,通过流式细胞术分选导致收集数千至数万个细胞,这些细胞通过制备,细胞收集方法(大量或单个细胞)和VCAM1富集而变化。&nbsp;

数据分析

  1. 请参阅BioRxiv上发布的Yousef et al。(2018)进行数据分析
    这个BEC隔离协议在Yousef 等人(2018)论文的以下图中使用:
    图1a-c:年轻和年老皮质/海马BEC的大量RNA-Seq。
    补充图1a-e:年轻和年老皮质/海马BEC的大量RNA-Seq。
    补充图1f-k: C57BL6小鼠在灌注前2小时注射抗VCAM1-DL488或IgG-DL488同种型对照(ro),以在脑解离,染色和FACS之前在体内标记BEC。 。然后将海马细胞用于单细胞RNA-Seq(图2和补充图2)。
    图2和补充图2:从海马分离的BEC的单细胞RNA-Seq。
    图3c-d:从用年轻或年老血浆处理的幼小鼠中分离BEC,并在灌注前2小时注射抗VCAM1-DL488或IgG-DL488同种型对照(ro)以标记BEC 在脑解离,染色和FACS之前的体内。用LPS刺激的幼鼠被用来设置门。&nbsp;
  2. 请参阅Czupalla et al。(2018) Bio-Protocol 中的“使用流式细胞仪对原代小鼠脑内皮细胞进行基于胶原酶的单细胞分离”的替代方案用于通过流式细胞术分离BEC细胞,其使用更温和的基于胶原酶的方法和几种标记物来染色其他细胞类型。该方案用于在BioRxiv上发表的Yousef 等人(2018)论文中的补充图7a-d,以显示在他莫昔芬处理的Vcam1 fl / fl <中的Vcam1的遗传重组和缺失。 / sup> Slco1c1-Cre ERT2 +/-小鼠。

食谱

  1. 改性DPBS(mDPBS)(500 ml)&nbsp;
    1. 在量筒中测量500mg葡萄糖
    2. 向量筒中加入1.36ml 100mM丙酮酸钠,然后加入DPBS至500ml切口
    3. 使用封口膜密封量筒,并通过倒置几次彻底混合
    4. 混合后,使用Thermo Scientific Nalgene Rapid-Flow无菌过滤装置(0.2μm孔)进行无菌过滤
  2. FACS缓冲液(500毫升)
    1. 将2.5g BSA和2ml 0.5M EDTA加入500ml mDPBS中
    2. 使用具有0.2μm孔的Thermo Scientific Nalgene Rapid-Flow无菌过滤器单元彻底混合并使用无菌过滤器
  3. 0.9 M蔗糖(500 ml)
    1. 在量筒中测量154.04g蔗糖
    2. 将DPBS添加至500毫升切口
    3. 使用封口膜密封量筒,并通过倒置几次彻底混合
    可选:如果混合物未完全溶解,请在加热的水浴中轻轻加热溶液并混合。混合后,使用Thermo Scientific Nalgene Rapid-Flow无菌过滤装置(0.2μm孔)进行无菌过滤。

致谢

我们感谢Lusijah Sutherland博士和Corey Cain博士,负责管理Palo Alto VA的核心流式细胞仪设施并提供H.Y.和C.J.C.关于文书的培训; Corey Cain博士还提供他的实验建议,流式细胞仪检测和PBMC分析以及深思熟虑的讨论。我们还要感谢Ryan Watts博士和Nga Bien-Ly博士分享用于RNA-Seq的原始BEC分离方案。这项工作由退伍军人事务部(TW-C。),国家老龄化研究所(SPO:116650; 1F32AG051330-01A1至HY,R01-AG045034和DP1-AG053015至TW-C)资助,NOMIS基金会(TW-C。),格伦老龄化研究基金会(TW-C),向HY提供SPARK资助通过斯坦福临床和转化科学奖(CTSA)到Spectrum(UL1 TR001085),美国国立卫生研究院(R01-GM37734和R37-AI047822到ECB,RO1 AI109452到HH),斯坦福免疫,移植和感染研究所( CJC)和Edinger Institute(CJC)。 CTSA计划由美国国立卫生研究院(NIH)的国家推进转化科学中心(NCATS)领导。

利益争夺

没有竞争利益冲突。

伦理

根据美国国立卫生研究院的实验动物护理和使用指南,在该方案中使用的小鼠在内部繁殖并老化并在无病原体条件下在12小时光 - 暗循环下生活。

参考

  1. Abbott,N.J.,Patabendige,A.A.,Dolman,D.E.,Yusof,S.R。和Begley,D.J。(2010)。 血脑屏障的结构和功能。 Neurobiol Dis 37(1):13-25。
  2. Banks,W。A.(2016)。 从血脑屏障到血脑界面:中枢神经系统药物输送的新机会。 Nat Rev Drug Discov 15(4):275-292。
  3. Czupalla,C.J.,Yousef,H.,Wyss-Coray,T。和Butcher,E.C。(2018)。使用流式细胞仪基于胶原酶的原代小鼠脑内皮细胞单细胞分离。 Bio-协议 8(22):e3092。
  4. Daneman,R.,Zhou,L.,Agalliu,D.,Cahoy,J.D.,Kaushal,A。和Barres,B.A。(2010)。 小鼠血脑屏障转录组:了解脑内皮细胞发育和功能的新资源。 PLoS One 5(10):e13741。
  5. Darmanis,S.,Sloan,S.A.,Zhang,Y.,Enge,M.,Caneda,C.,Shuer,L。M.,Hayden Gephart,M。G.,Barres,B。A.和Quake,S。R.(2015)。 单细胞水平的人脑转录组多样性调查。 Proc Natl Acad Sci USA 112(23):7285-7290。
  6. Engelhardt,B.,Vajkoczy,P。和Weller,R。O.(2017)。 中枢神经系统免疫特权的推动者和塑造者。 Nat Immunol < / em> 18(2):123-131。
  7. Picelli,S.,Faridani,O.R.,Bjorklund,A.K.,Winberg,G.,Sagasser,S。和Sandberg,R。(2014)。 使用Smart-seq2从单个细胞中获得全长RNA-seq。 Nat Protoc 9(1):171-181。
  8. Tam,S.J.,Richmond,D.L.,Kaminker,J.S.,Modrusan,Z.,Martin-McNulty,B.,Cao,T.C.,Weimer,R.M.,Carano,R.A.,van Bruggen,N。和Watts,R.J。(2012)。 死亡受体DR6和TROY调节脑血管发育。 Dev Cell 22(2):403-417。
  9. Vanlandewijck,M.,He,L.,Mae,MA,Andrae,J.,Ando,K.,Del Gaudio,F.,Nahar,K.,Lebouvier,T.,Lavina,B.,Gouveia,L。, Sun,Y.,Raschperger,E.,Rasanen,M.,Zarb,Y.,Mochizuki,N.,Keller,A.,Lendahl,U。和Betsholtz,C。(2018)。 脑血管系统中细胞类型和分区的分子图谱。 自然 554(7693):475-480。
  10. Yousef,H.,Czupalla,CJ,Lee,D.,Burke,A.,Chen,M.,Zandstra,J.,Berber,E.,Lehallier,B.,Mathur,V.,Nair,RV,Bonanno, L.,Merkel,T.,Schwaninger,M.,Quake,S.,Butcher,EC和Wyss-Coray,T。(2018)。 老年血液抑制海马神经发生,并通过血脑屏障的VCAM1激活小胶质细胞。 bioRxiv 。 doi:10.1101 / 242198。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yousef, H., Czupalla, C. J., Lee, D., Butcher, E. C. and Wyss-Coray, T. (2018). Papain-based Single Cell Isolation of Primary Murine Brain Endothelial Cells Using Flow Cytometry. Bio-protocol 8(22): e3091. DOI: 10.21769/BioProtoc.3091.
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