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Jan 2018

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Collagenase-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胶原酶的单细胞分离   

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Abstract

The brain endothelium is a highly specialized vascular structure that maintains the activity and integrity of the central nervous system (CNS). Previous studies have reported that the integrity of the brain endothelium is compromised in a plethora of neuropathologies. Therefore, it is of particular interest to establish a method that enables researchers to investigate and understand the molecular changes in CNS endothelial cells and underlying mechanisms in conjunction with murine models of disease. In the past, approaches to isolate endothelial cells have either involved the use of transgenic reporter mice or suffered from insufficiently pure cell populations and poor yield.

This protocol here is based on well-established protocols that were modified and combined to allow single cell isolation of highly pure brain endothelial cell populations using fluorescence activated cell sorting (FACS). Briefly, after careful removal of the meninges and dissection of the cortex/hippocampus, the brain tissue is mechanically homogenized and enzymatically digested in two steps resulting in a single cell suspension. Cells are stained with a cocktail of fluorochrome-conjugated antibodies identifying not only brain endothelial cells, but also potentially contaminating cell types such as pericytes, astrocytes, and lineage cells. Using flow cytometry, cell populations are separated and sorted directly into either RNA lysis buffer for bulk RNA analyses (e.g., RNA microarray and RNA-Seq) or in pure fetal bovine serum to preserve viability for other downstream applications such as single cell RNA-Seq and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq). 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 3 x 105 cells from a pool of four adult mice.

Keywords: CNS (CNS), Brain endothelium (脑内皮细胞), Pericytes (周细胞), Blood-brain barrier (血脑屏障), Neuro-vascular unit (神经血管单元), Single cell isolation (单细胞分离), RNA-Seq (RNA-Seq), RNA microarray (RNA微矩阵)

Background

The brain endothelium serves as an interface for systemic factors circulating through the blood. Cerebral capillary endothelial cells constitute the blood-brain barrier (BBB), a physical barrier that limits paracellular flux via unique tight junction protein formations interconnecting cells, to maintain homeostasis of the neuronal environment and thus, neuronal functionality (Liebner et al., 2011 and 2018). The BBB not only limits the passage of ions and other molecules such as glucose but also prevents uncontrolled exchange of toxins, bacteria, viruses and cells between the blood and the brain parenchyma (Abbott et al., 2010). To accomplish this task, the brain endothelium is dependent on a fine-tuned microenvironment within the neuro-vascular unit (NVU), which consist of endothelial cells with closely associated pericytes and astrocytes as well as extracellular matrix components and microglia (Abbott and Friedman, 2012).

The function and/or integrity of the BBB is compromised in several neuropathologies, such as Alzheimer’s disease (AD), multiple sclerosis, epilepsy, and glioblastoma (Zlokovic, 2008; Marchi et al., 2012; Wolburg et al., 2012). The fluorescent activated cell sorting (FACS) single cell isolation method described here has been developed in the context of a study of transcriptional changes in brain endothelial cells in AD and inflammation (unpublished). It is a modification of published protocols for brain endothelial cell cultivation (Liebner et al., 2008; Czupalla et al., 2014), and is designed to dissociate brain EC into a single cell suspension while preserving many endothelial surface antigens for flow cytometry. We have successfully applied the method in a collaborative investigation of the effects of an aged systemic milieu on hippocampal neurogenesis and microglia activation and the role of vascular cell adhesion molecule 1 (VCAM-1) as a negative regulator of adult neurogenesis and inducer of microglial activity (Yousef et al., 2018a). For an alternative brain endothelial cell isolation protocol–slightly faster, but utilizing less gentle enzymatic digestion–see Yousef et al. (2018b).

In the last decade, several protocols for brain endothelial isolation have been employed predominantly in BBB developmental studies (Daneman et al., 2010; Vanlandewijck et al., 2018). However, these techniques are dependent on the presence of transgenic endothelial cell markers and thus, may not easily be applied to transgenic disease mouse models. In addition, depending on the digestion enzyme used, epitopes essential for brain endothelial cell detection, such as CD31 are destroyed. This protocol allows the isolation of highly pure brain endothelial cell population (and pericytes) from any mouse strain or murine animal model using gentle enzymatic digestion steps that preserve endothelial-specific epitopes.

Materials and Reagents

  1. Falcon 15 ml conical centrifuge tubes (Fisher Scientific, Corning, catalog number: 352096)
  2. Falcon 50 ml conical centrifuge tubes (Fisher Scientific, Corning, catalog number: 352070)
  3. Bottle-top vacuum filters, pore size 0.45 μm (MilliporeSigma, Corning, catalog number: CLS430514-12EA) 
  4. Bottle-top vacuum filters, pore size 0.22 μm (MilliporeSigma, Corning, catalog number: CLS430513-12EA) 
  5. Disposable sterile bottles (Fisher Scientific, Corning, catalog number: 09-761-10)
  6. 1.5 ml Snap-Cap Microcentrifuge Safe-LockTM Tubes (Eppendorf, catalog number: 022363204)
  7. 1 ml Insulin Syringe with Slip Tip (BD, catalog number: 329654)
  8. 1 ml Millex-GV Filter, 0.22 µm (MilliporeSigma, catalog number: SLGV004SL)
  9. Petri Dish 100 x 21 mm (Thermo Fisher Scientific, NuncTM, catalog number: 172931)
  10. Petri Dish 60 x 15 mm (Thermo Fisher Scientific, NuncTM, catalog number: 150326)
  11. Serological pipette 10 ml, sterile (SARSTEDT, catalog number: 86.1254.025)
  12. Optional: 5 ml conical tubes (Eppendorf, catalog number: 0030119487) (see Notes)
  13. Serological pipette 5 ml, sterile (SARSTEDT, catalog number: 86.1253.025)
  14. 5 ml Test Tube with Cell Strainer Snap Cap (Falcon, catalog number: 352235)
  15. Disposable Borosilicate Glass Pasteur Pipets, autoclaved (Fisher Scientific, catalog number: 13-678-20C)
  16. Screw-Cap microcentrifuge tubes, 1.5 ml (VWR, catalog number: 89004-290)
  17. Glass or plastic Beaker, 1,000 ml and 200 ml (laboratory specific)
  18. Autoclaved WhatmanTM qualitative filter paper, Grade CF 12 (Sigma-Aldrich, catalog number: WHA10535097) (see Notes)
  19. WhatmanTM Grade 1573-1/2 Qualitative Filter Papers (GE Healthcare, catalog number: 09-927-210)
  20. Disposable scalpel blades, sterile (IntegraTM Miltex®, catalog number: 4-123)
  21. Adult mice, 6-12 weeks, e.g., C57/Bl6 (The Jackson Laboratory, catalog number: 000664)
  22. Deionized, filtered water (dH2O) (Merck Millipore, Milli-Q)
  23. Bovine Serum Albumin, Fraction V, Heat Shock Treated (BSA) (Fisher Scientific, Fisher BioReagentsTM, catalog number: BP1600-1)
  24. Sodium chloride (NaCl) (MilliporeSigma, catalog number: S3014)
  25. Potassium chloride (KCl) (MilliporeSigma, catalog number: PX1405)
  26. Calcium chloride (CaCl2) (MilliporeSigma catalog number: 102391)
  27. Magnesium chloride (MgCl2) (MilliporeSigma catalog number: M8266)
  28. HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080)
  29. Sodium hydroxide (NaOH), pellets (MilliporeSigma catalog number: S8045)
  30. Water, Molecular Biology Reagent (MilliporeSigma, catalog number: W4502-1L)
  31. Ethanol 70% W/V (MilliporeSigma, catalog number: EX0281-1)
  32. Red Blood Cell Lysis Buffer (MilliporeSigma, catalog number: 11814389001)
  33. Sodium phosphate monobasic (NaH2PO4) (MilliporeSigma, catalog number: S3139)
  34. Potassium phosphate dibasic (KH2PO4) (MilliporeSigma, catalog number: S3264)
  35. Fetal Bovine Serum (FBS), Defined (HyCloneTM, catalog number: SH30070.03)
  36. Hanks' Balanced Salt Solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 24020117)
  37. CD31 (PECAM-1) Monoclonal Antibody (390), PE-Cyanine7 (eBioscience, catalog number: 25-0311-82)
  38. MECA-99 (Eugene C. Butcher Laboratory) (Kruse et al., 1999; Lee et al., 2014)
  39. DyLightTM 488 Antibody Labeling Kit (Thermo Fisher Scientific, catalog number: 53024)
  40. Aminopeptidase N/CD13 Antibody (ER-BMDM1) (NOVUS Biologicals, catalog number: NB100-64843)
  41. ACSA-2 (Miltenyi, catalog number: 130-097-674)
  42. Anti-ACSA-2-PE, mouse (Miltenyi, catalog number: 130-102-365) 
  43. Anti-Mouse CD45 PerCP-Cy5.5 (eBioscience, catalog number: 45-0451-82)
  44. Anti-mouse CD11a/CD18 (LFA-1) PerCP/Cy5.5 (BioLegend, catalog number: 141008)
  45. Anti-Mouse CD11b PerCP-Cyanine5.5 (eBioscience, catalog number: 45-0112-80)
  46. Anti-Mouse TER-119 PerCP-Cyanine5.5 (eBioscience, catalog number: 45-5921-82)
  47. UltraComp eBeadsTM (eBioscience, catalog number: 01-2222-42)
  48. Propidium iodide solution (PI) (MilliporeSigma, catalog number: P4864)
  49. Optional: RNeasy Plus Micro Kit (50) (QIAGEN, catalog number: 74034)
  50. Optional: Array, Mouse Gene 1.0 ST ARRAY (Affymetrix, catalog number: 901169)
  51. Collagenase type II (Biochrom Kg, catalog number: C2-22)
  52. Collagenase/Dispase (MilliporeSigma, catalog number: 11097113001)
  53. Deoxyribonuclease I (CellSystems, catalog number: LS006331)
  54. Stock solutions (see Recipes)
  55. PBS for BSA (see Recipes)
  56. Endothelial cell buffer (see Recipes)
  57. Collagenase II solution (see Recipes)
  58. 25% BSA (see Recipes)
  59. Collagenase/Dispase solution (see Recipes)
  60. DNase I solution (see Recipes)
  61. FACS Buffer (see Recipes)
  62. Antibody dilutions (see Recipes)

Equipment

  1. Scale and weigh supplies (Laboratory-specific)
  2. DURAN® Erlenmeyer flask, 2,000 ml (DURAN, catalog number: 21 216 63)
  3. -20 °C freezer (laboratory-specific)
  4. Precision GP 10 L General Purpose Water Bath (Precision Scientific, catalog number: TSGP10)
  5. Portable Pipet-Aid® XP Pipette Controller (DRUMMOND, catalog number: 4-000-101)
  6. Thermo ScientificTM NalgeneTM Polypropylene Powder Funnels (Thermo ScientificTM, catalog number: 42520150)
  7. Cylinder, Cylinder, Grad. Cls B, 1,000 ml and 100 ml
  8. Magnetic Stirrer (Thermo Fisher Scientific, catalog number: 90-691-18)
  9. FisherbrandTM Round Stir Bars with Removable Pivot Ring (Thermo Fisher Scientific, fit size to measure column)
  10. FisherbrandTM accumetTM AB15 Basic and BioBasicTM pH/mV/°C Meters or other pH meter
  11. Autoclave (laboratory-specific)
  12. Carbon dioxide chamber (laboratory-specific)
  13. Thermo-isolated container including a lid, filled with ice
  14. Surgical Scissors, Sharp (Fine Science Tools, catalog number: 14002-16)
  15. Fine Scissors, Blunt-Blunt (Fine Science Tools, catalog number: 14108-09)
  16. Semken Forceps, Straight (Fine Science Tools, catalog number: 11008-13)
  17. GSC Go Science Crazy Stainless-Steel Spatula (Fisher Scientific, catalog number: S50788A)
  18. Dumont #3 Forceps (Fine Science Tools, catalog number: 11293-00)
  19. Dumont #5–Ceramic Coated Forceps (Fine Science Tools, catalog number: 11252-50)
  20. Scalpel Handle #4 (Fine Science Tools, catalog number: 10004-13)
  21. Vacuum pump (laboratory-specific)
  22. Centrifuge 5810 R swing bucket for conical tubes (Eppendorf, model: 5810 R)
  23. PIPETMAN Classic P2 (Gilson, catalog number: F144801) 
  24. PIPETMAN Classic P10 (Gilson, catalog number: F144802) 
  25. PIPETMAN Classic P100 (Gilson, catalog number: F123615) 
  26. PIPETMAN Classic P200 (Gilson, catalog number: F123601) 
  27. PIPETMAN Classic P1000 (Gilson, catalog number: F123602)
  28. 4 °C fridge (laboratory-specific)
  29. Nikon SMZ 745, Stereomicroscope (Nikon, model: SMZ 745)
  30. BD FACSAriaTM II or III cell sorter (BD Biosciences)

Software

  1. BD FACSDIVATM SOFTWARE (BD Biosciences, version: V8.0.1)
  2. FlowJoTM (© FlowJo, LLC, version: 9.9.4 or higher)
  3. Optional: GeneSpring GX (Agilent Technologies, version: 14.8)
  4. Optional: Partek® Genomics Suite® (Partek Incorporated)

Procedure

Notes:

  1. Figure 1 outlines the workflow of the procedure to provide an overview of the individual steps.
  2. This protocol does not require a sterile technique, but sterile equipment and reagents are used whenever possible, to minimize potential contamination and foster cell viability.
  3. Sterilize dissection tools with 70% (vol/vol) ethanol (glass beaker). Let tools dry off before use.
  4. Pre-chill endothelial cell buffer (see Recipes) and 6 cm Petri dish on ice.
  5. Thaw 25% Bovine Serum Albumin (BSA) (see Recipes) in a 37 °C water bath, mix well by gently inverting the tube and immediately put on ice when thawed.
  6. Thaw and pre-warm 2,400 U/ml collagenase II (see Recipes) in a 37 °C water bath for 20 min before use.
  7. Cool centrifuge to 4 °C.
  8. The yield of this protocol is highly dependent on the correct choice of volumes and tube sizes. The protocol described below is designed for isolating cells from a pool of four adult mouse brains (see Notes). For additional brains/experimental groups add extra sample tubes to the process. 


    Figure 1. Protocol overview for purification of murine brain endothelial cells isolated from cortices/hippocampi of adult mice. For details refer to Procedure A-H as indicated in this workflow chart. The image contains graphic elements from Motifolio toolkit.


  1. Brain tissue harvest
    1. Sacrifice adult mice using carbon dioxide asphyxiation followed by cervical dislocation (adhere to institutional guidelines). Harvest up to four brains at a time.
    2. Spray the head/neck area with 70% (vol/vol) ethanol and remove the head from the torso using sharp surgical scissors (Figure 2). 
    3. Carefully dissect the brain from the skull at room temperature. It is critical to preserve the integrity of the brain tissue, as cutting into or puncturing the tissue may lead to difficulties during the meningeal removal and the cortex/hippocampi dissection. Use blunt scissors to cut open the skull along the left and right side (enter through the spinal cord extension), while pushing the tip of the scissors gently outwards (Figure 2). 
    4. Carefully lift the upper part of the skull few millimeters and make another cut half way through the middle of the skull and strip away the skull pieces side using forceps (Figure 2). 
    5. Use the small spatula to remove the brain from the skull (Figure 2).
    6. Place brains in a 6 cm Petri dish on ice and add ice cold endothelial cell buffer to cover (Figure 2).


      Figure 2. Brain tissue harvest. Illustration of the brain tissue harvest procedure described in Procedure A (adult wild-type Balb/c mouse). Step 1 is animal sacrifice.

    Notes: 
    1. From here on, process only one brain at a time until noted otherwise. 
    2. Ensure that the dissection process described in this section does not take longer than 15 min per brain. While practicing the technique, chill tissue covered in endothelial cell buffer on ice in-between steps to preserve cell viability. Alternatively, tissue dissection may be performed in a Petri dish on ice if the microscope setup allows. However, meninges will be easier to identify on a dark background.

  2. Cortex/hippocampi dissection
    1. Transfer the brain to an empty 10 cm Petri dish to remove the cerebellum (including pons and medulla if applicable) and the olfactory bulb using a scalpel, while carefully stabilizing the brain with forceps.
    2. Place the brain onto an autoclaved sheet of Whatman filter paper and gently roll over the surface using a sterile spatula to crudely remove the meningeal layers surrounding the brain (Figure 3). Since brain tissue is soft and sticky, ensure to carefully separate it from the filter paper using a small spatula during rolling to minimize tissue loss. Cortical/hippocampal microvessels have distinct phenotypical properties that differ from those of meningeal vessels and inclusion of the later results in contamination with undesired endothelial cell populations.
    3. Use the small spatula (not forceps) to transfer the tissue onto a 10 cm Petri dish and cover it with ice cold endothelial cell buffer.
    4. Place the dish under a binocular microscope and continue to manually remove the remaining meninges using Ceramic Coated Dumont #5 forceps, while stabilizing the tissue with another set of forceps (Dumont #3 or #5) (Figure 3). Despite the fact that the meninges may be hard to see, as they are delicate and mostly transparent, aim to remove all meningeal structures. Pay particular attention to “red-colored” tissue areas, as they indicate erythrocyte-filled blood vessels, which form a network throughout the meningeal layers and therefore serve as an indicator as to which areas have to be cleaned from meninges.
    5. Separate the hemispheres with a clean cut along the interhemispheric fissure using a sterile scalpel blade (Figure 3).
    6. Transfer one hemisphere into a Petri dish with endothelial cell buffer on ice and continue to dissect the cortex and hippocampus of the remaining half.
    7. Grab a small area of the “whitish” brain tissue–previously enclosed by the cortices including midbrain, thalamus, hypothalamus, basal forebrain, caudate putamen, ventral striatum, anterior olfactory nucleus, and other brain regions not listed here–in the posterior region with Dumont forceps #3 or #5. Then stab onto the tissue below in an almost parallel angle to the bench top, to cause incision using the Dumont forceps #5 like scissors (Figure 3). Keep cutting along the slightly more “pinkish” cortex until the undesired “whitish” tissue is removed. Ideally, you see the rim surface of the cortex and the embedded hippocampus after this dissection (Figure 3). However, if this is not the case, continue to remove undesired tissue parts piece by piece. 
    8. Continue to remove meninges in the medial longitudinal fissure and flip over the hemisphere to thoroughly inspect and clean-up the other side of the cortex as well.
    9. Transfer cortex/hippocampus to a clean Petri dish on ice filled with endothelial cell buffer.
    10. Go through Steps B6 to B9 for the second hemisphere and thereafter start over at Step B1 with the next brain until all brains have been processed (~two hours total for 2 x 4 brains; do not exceed 3 h for this part of the procedure, as cell viability will be affected).
    11. Thereafter, process pooled tissue.


      Figure 3. Selected steps of the cortex/hippocampus dissection described in Procedure B. Bottom right image illustrates the location of the hippocampus indicated by the green dotted line. 

  3. Tissue homogenization and primary digestion 
    1. Wash the pooled brain tissue three times by repeatedly adding endothelial cell buffer, swirling the Petri dish, followed by careful aspiration using a vacuum pump.
    2. After the last aspiration, mince the tissue using two sterile scalpels into small pieces until a homogenous emulsion has formed.
    3. Add 4 ml endothelial cell buffer to the Petri dish and resuspend the tissue by pipetting up and down with a 10 ml pipette. 
    4. Transfer the cell suspension into a 15 ml Falcon tube kept on ice.
    5. Repeat Steps C3 and C4 two more times to rinse the Petri dish and top off with endothelial cell buffer to 15 ml.
    6. Centrifuge cell suspension for 7 min at 300 x g at 4 °C and aspirate the supernatant using a vacuum pump. 
    7. Estimate the volume of your tissue pellet (typically ~1.5 ml) and add endothelial cell buffer as well as pre-warmed collagenase II in a 1:1:1 ratio. Pipette gently up and down using a 5 ml pipette.
    8. Incubate the enzyme mixture in a 37 °C water bath for 50 min and thoroughly shake the tube after 25 and 50 min of incubation to homogenize the suspension until no white clumps are visible.
    9. Stop the enzymatic digestion (~4.5 ml volume) by adding endothelial cell buffer to 15 ml and mix suspension by thoroughly pipetting up and down.
    10. Centrifuge cell suspension for 7 min at 300 x g at 4 °C and aspirate the supernatant using a vacuum pump. 

  4. Myelin removal and erythrocyte depletion
    1. Add 3 ml 25% BSA and transfer to a new 15 ml tube. Rinse the original tube one more time with 3 ml 25% BSA and then top off to 15 ml, thoroughly mixing the suspension with a 10 ml pipette.
    2. Centrifuge for 30 min at 1,000 x g at 4 °C in order to separate the myelin (top) and to enrich for capillary fragments (bottom) (Figure 1D).
    3. Aspirate the myelin layer with a vacuum pump. Before removing the clear BSA supernatant, switch to a new tip to minimize residual myelin in the cell pellet.
    4. To deplete erythrocytes, incubate the pellet in 2 ml Red Blood Cell Lysis Buffer for 90 s at room temperature with occasional shaking. Add 1 ml Red Blood Cell Lysis Buffer with a P1000 pipette, transfer suspension to a new 15 ml Falcon tube, and rinse the tube one more time with Red Blood Cell Lysis Buffer 1 ml and combine. 
    5. Inhibit cell lysis by adding 13 ml endothelial cell buffer and put sample back on ice.
    6. Centrifuge cell suspension for 7 min at 300 x g at 4 °C and aspirate the supernatant using a vacuum pump and leave ~2 ml in the tube. Use a 1 ml pipette to carefully remove the remaining supernatant.

  5. Secondary digestion–single cell suspension
    1. Resuspend the cell pellet in 2 ml endothelial cell buffer and transfer cell suspension to a new 15 ml Falcon tube.
    2. To digest the microvessel fragments into a single cell suspension, add 1 mg/ml Collagenase/Dispase and incubate the mixture in a 37 °C water bath for 13 min.
    3. Notice the formation of endothelial microvessel fragment aggregates clustered by DNA. Add 1 μg/ml DNase I, pipette up and down a few times until the microvessels are dissociated using a P1000 pipette, and incubate for an additional 2 min in the 37 °C water bath. 
    4. To quench the digestion reaction, add 13 ml endothelial cell buffer and mix by gently inverting the tube (do not pipette up and down).
    5. Centrifuge cell suspension for 10 min at 300 x g at 4 °C.
    6. Aspirate the supernatant using a vacuum pump and leave ~2 ml in the tube. Use a 1 ml pipette to carefully remove the remaining supernatant and store cell pellet on ice.
    7. Resuspend the pelleted cells in FACS buffer (see Recipes). The total resuspension volume should be 200 μl per antibody cocktail FACS sample plus 50 µl for the unstained control (for additional controls see Notes section below). 
    8. Distribute the resuspended cells into appropriately labeled 1.5 ml tubes for the antibody cocktail FACS sample (200 µl) and the unstained control (50 µl) and store on ice.
    Notes: 
    1. Important controls for FACS parameter setup include single-color positive controls, to compensate for channel spillover. Since the sample cell numbers are a limiting factor, we recommend using compensation beads in combination with an unstained cell sample control to set up forward and side scatter.
    2. We further recommend to establish proper gating using FMO (fluorescence minus one) controls, in which cells are stained with all antibodies except one. This is particularly important for initial FACS setup and should be repeated when using new antibody batches, as signal intensity may vary from batch to batch. For detailed information see Tung et al. (2007). 
    3. Plan to run an experiment just for establishing these settings on your FACS device. In our hands, the procedure is highly reproducible and saved experimental settings with minor adjustments may be used for subsequent experiments, maximizing the available cell sample for sorting. 

  6. Immunostaining for FACS
    1. Label 5 ml FACS test tubes for each fluorochrome used in the experiment plus one for unstained control and add 150 µl FACS buffer per tube (compensation controls).
    2. Mix UltraComp eBeads by vigorously inverting at least 10 times and add three drops to a 1.5 ml tube and add 100 µl FACS buffer.
    3. Apply 50 µl of the diluted compensation bead dilution to each compensation control tube.
    4. Add antibodies at the same dilution (see Recipes) used for FACS sample staining (chose one antibody for PerCP-Cy5.5).
    5. Mix well by flicking the tube and incubate on ice for 20 min in the dark.
    6. Add all antibodies as indicated to the antibody cocktail FACS sample (see Recipes) and carefully mix by pipetting up and down using a P100 pipette (avoid bubbles).
    7. Incubate on ice for 30 min in the dark.
    8. During the incubation time, prepare collection tubes for desired downstream application:
      1. RNA extraction: prepare two 1.5 ml screw cap tubes with 750 µl RLT Plus lysis buffer each (see manufacturer guidelines). Ensure that the volume is not significantly lower than 750 µl as a ratio lower than 1:1 of lysis buffer to sorted sample may result in reduced RNA quality. Keep collection tubes at room temperature.
      2. Intact cells: prepare two 1.5 ml screw cap tubes with 500 µl FBS each and chill on ice.
    9. Wash off excess antibodies by adding 1 ml FACS buffer to each tube (FACS sample as well as compensation beads) and centrifuge for 5 min at 300 x g at 4 °C.
    10. Decant supernatant of compensation beads tubes.
    11. Carefully aspirate FACS sample supernatant using a P1000 and then P200 pipette. 
    12. Resuspend FACS sample pellet in 300 µl and compensation beads in 200 µl FACS buffer.
    13. Keep compensation controls on ice and in the dark until FACS.
    14. Add 150 µl FACS buffer to the unstained cell sample kept on ice and mix well as above. 
    15. Ensure to dissociate potential sample cell aggregates using a P200 pipette before transferring the FACS samples onto the blue filter cap of a 5 ml Test Tube with Cell Strainer.
    16. Spin cells through the filter for 2 min at 300 x g at 4 °C and store samples on ice in the dark.
    17. Transport samples and a 1:10 propidium iodide (PI) pre-dilution (in FACS buffer) to the FACS facility. Keep cells on ice and in the dark at all times.
    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. 
    5. 10%-25% of total events are CD31/MECA-99+ brain endothelial cells; sorted brain endothelial cell count ranges from 1-5 x 105 cells; average count from four adult mice: 3 x 105 cells.

  7. FACS and sample collection
    1. Run unstained cells to set up forward (FSC-A) and side scatter (SSC-A) (Figure 4).
    2. Run single-color controls to set FACS parameters and compensate for channel spillover.
    3. Add 1:3,000 (1:300 from 1:10 pre-dilution; equals 0.33 µg/ml) PI for live/dead staining right before running the individual sample, as it affects cell viability.
    4. Run FMO controls for new batches of antibody, to determine positive cell populations and set up gates accordingly (Figure 4). For more detailed information see Tung et al. (2007).


      Figure 4. FACS gating strategy for purification of single cells endothelial cells. A. Cells are gated on forward (FSC-A = size) and side scatter (SSC-A = internal structure) to exclude cell debris and residual myelin. B. FSC-A and FSC-W plotting was used to discriminate single cells from cell doublets/aggregates. C. PI uptake indicates dead cells, which are excluded. D. CD45, CD11a/b, and Ter-119 negative cells are gated to exclude erythrocytes, monocytes/macrophages, and microglia. E. CD13 and ACSA-2 staining is applied to exclude pericytes (orange) and astrocytes (red), respectively. The CD13 positive population consists of pericytes and pericyte/endothelial cell doublets (orange) and may be further gated on both endothelial markers CD31/MECA-99 (not shown), resulting in the double negative population to represent pericytes. F. CD31/MECA-99 double positive cells are defined as the brain endothelial cell population. The blue numbers indicate the event percentage of the parental gate. Dot plots show 50k recoded events.

    5. Run stained FACS sample
      1. Gate cells on forward (FSC-A = size) and side scatter (SSC-A = internal structure) to exclude cell debris and residual myelin (Figure 4A).
      2. Plot FSC-A against FSC-W to discriminate single cells from cell doublets/aggregates (Figure 4B). For more detailed information see Tung et al. (2007).
      3. Exclude PI positive (dead) cells (Figure 4C).
      4. Exclude erythrocytes, monocytes/macrophages, and microglia by gating on CD45, CD11a/b, and Ter-119 negative cells (Figure 4D).
      5. Exclude pericytes (CD13+) and astrocytes (ACSA-2+) by gating on the double negative population (Figure 4E) (see Notes).
      6. Gate on CD31/MECA-99 double positive cells and collect cells from this gate to obtain a pure brain endothelial cell population (Figure 4F). Record 1 million events.
    6. Collect cells in 1.5 ml screw-cap microcentrifuge tubes containing (a) RLT Plus lysis buffer, for direct cell lysis and bulk RNA downstream applications or (b) FBS, for downstream applications requiring intact/single cells. 

  8. Selection of optional downstream applications
    1. RNA microarray (Sealfon and Chu, 2011) (Figure 5).
    2. RNA-Seq, bulk (Kukurba and Montgomery, 2015).
    3. Single cell RNA-Seq (Haque et al., 2017).
    4. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq) (Buenrostro et al., 2013 and 2015).
    5. We do not recommend cultivation of sorted endothelial cells as the procedure causes stress to the cells that impacts expression profiles. Cells may attach to extracellular matrix-coated surfaces but won’t proliferate sufficiently. 


      Figure 5. Quality control of FACS sorted cell populations from the murine brain. A-D. Microvascular tubes (MVT) were isolated (Fisher et al., 2007) and seeded on fibronectin-coated glass slides for double immunohistochemistry staining of brain endothelial marker CD31, pericyte marker Desmin, and astrocytic markers GFAP and Aqp4 indicating insufficient cell separation using mechanical tissue dissociation. Black scale 50 µm and white scales 25 µm. MVTs served as mixed cell population control for RNA microarray studies (E-G). E. Principal-component analysis (PCA) of the transcriptomes of brain capillary endothelial cell (CAP), pericyte (PC), astrocyte (AC), and microglia (MG) single cell populations isolated using flow cytometry. For purification of CAP and PC populations, the outlined protocol was applied, while ACs and MGs were isolated using an unpublished method. PCA was calculated for normalized EVs with a difference of at least two-fold between any pair of samples (P < 0.05 [one-way ANOVA]) and with raw expression (EV) of >140 in 100% of replicates of at least one sample population. Numbers in parentheses indicate the proportion of total variability calculated for each principal component (PC). Each data point represents a biological replicate of cells sorted from tissues pooled from 2-8 adult C57/Bl6 mice. Analysis implicates well-defined cell populations clustering together based on their cell type-specific transcriptomes. F. Hierarchical clustering by correlation of samples MVT, CAP, PC, AC, and MG with the gene list defined in E based on log2 normalized EVs. Each terminal branch represents results from a single microarray analysis of an independent biological replicate of sorted cells as described in E. G. Hierarchical clustering by correlation of selected cell-type defining gene markers: Aqp4, GFAP, and Acsa-2 (AC); Pdgfrb, CD13, and Desmin (PC); CD45, CD11b, Iba-1, and CD68 (MG); Tie2, Podxl, Cldn5, and CD31 (CAP). Individual cell types cluster together and express high levels of known cell type-specific genes indicating highly pure cell populations. As a control, arrays of MVT show the presence of pericyte, endothelial, MG and astrocyte genes. 

Data analysis

The data analysis described below refers to purification quality control transcriptome analysis using RNA microarray technology shown in Figure 5. For purification of brain endothelial and pericyte populations the outlined protocol was applied, while microvascular tubes, astrocytes, and microglia were isolated using different techniques (Fisher et al., 2007 and Fisher et al., unpublished).

  1. RNA sample quality and quantity were determined by Agilent Bioanalyzer Total RNA Pico Euk chip readings at the Stanford University PAN facility.
  2. Three to fifty nanogram of total RNA, with a minimum RNA integrity number (RIN) of 8, was used for amplification, labeling, and hybridization performed by Expression Analysis (Q2Solutions). Samples were hybridized on Affymetrix Genechip Mouse Gene 1.0 ST arrays. 
  3. GeneSpring GX sample preprocessing and default normalization (RMA-16) were applied. Dynamic ranges of all RNA samples were at least 40.
  4. GeneSpring GX and Partek Genomic Suite software were used for processing and analysis of the transcriptome data. Principal-component analysis (PCA) was calculated for normalized EVs with a difference of at least two-fold between any pair of samples (P < 0.05 [one-way ANOVA], permeative 100 multiple testing, Benjamini-Hochberg correction) and with raw expression (EV) of >140 in 100% of replicates of at least one sample population. 
  5. Each PCA data point represents a biological replicate of cells sorted from tissues pooled from 2-8 adult C57/Bl6 mice (ages: 6-14 weeks [cells] and 4 months [MVTs]).
  6. Hierarchical clustering was performed by correlation of all samples with the gene list defined in the PCA analysis based on log2 normalized EVs (Figure 5F). Each terminal branch represents results from a single microarray analysis of an independent biological replicate of sorted cells.
  7. Hierarchical clustering was performed by correlation of selected cell-type defining gene markers: Aqp4, GFAP, and Acsa-2 (astrocytes); Pdgfrb, CD13, and Desmin (pericytes); CD45, CD11b, Iba-1, and CD68 (microglia); Tie2, Podxl, Cldn5, and CD31 (brain capillary endothelial cells). 

Notes

  1. This protocol may be applied to brain tissue derived from adult mice of older ages as well. Be advised that the yield may decline with increasing age or may potentially be affected by transgene expression.
  2. This protocol may also be applied to individual brains. Use 5 ml instead of 15 ml tubes and 1.5 ml conical tubes as applicable and scale down the buffer and enzyme solution volumes (e.g., for one brain, use a quarter of the volumes indicated; incubation times remain the same).
  3. Cut Whatman filter paper sheet to an appropriate size (e.g., 18 x 12 cm) and autoclave wrapped in aluminum foil or autoclavable container.
  4. Ensure that the brain tissue stays moistened while removing meninges and during tissue dissection.
  5. Due to high fat content, brain tissue pellets are rather unstable and viscous. Use conical tubes at all times and make sure to use a swing bucket centrifuge to collect cells on the bottom of the tube. Never decant supernatant, but carefully aspirate using a vacuum pump for big volumes and a handheld pipettor for small volumes.
  6. Antibody concentrations are recommendations and should be tested since staining intensity might vary from batch to batch.
  7. The CD13+ pericyte population consists of individual pericytes as well as of pericyte/endothelial cell doublets. CD13+ cells may be further gated on both endothelial markers (not shown). The resulting CD31/MECA-99 double negative population represent pericytes. It is of note that the yield of pure pericytes is rather low (1-1.5 x 104) and requires more starting material, typically up to eight mice.
  8. MECA-99 antibody was produced and conjugated to DL488 in-house. You may choose a different conjugate if you wish to extend the fluorochrome panel by an additional color. E.g., in one of our studies, we injected VCAM-1-DL488 in vivo and stained cells with MECA-99-DL405 (Yousef et al., 2018a).
  9. RNA samples should not be put on ice but instead be processed right after sorting is finished. Alternatively, freeze RNA samples in RLP Plus buffer at -80 °C for short-term storage (up to four weeks).

Recipes

  1. Stock solutions (in dH2O)
    1. 2 M NaOH (50 ml)
    2. 5 M NaCl (100 ml)
    3. 1 M KCl (50 ml)
    4. 1 M CaCl2 (50 ml)
    5. 1 M MgCl2 (50 ml)
    6. 1 M Na2HPO4 (50 ml)
    7. 1 M KH2PO4 (50 ml)
    Notes: 
    1. Stock solutions b-g are for making endothelial cell buffer and PBS for BSA solution.
    2. Aliquot in 50 ml Falcon tubes and store at room temperature (check for precipitate before use and prepare fresh as required).
  2. PBS for BSA (combine all components in a 1,000 ml cylinder)
    1. 140 mM NaCl: 28.4 ml/5 M solution
    2. 0.2 mM CaCl2: 0.2 ml/1 M solution
    3. 0.2 mM MgCl2: 0.2 ml/1 M solution
    4. 10 mM Na2HPO4: 10 ml/1 M solution
    5. 10 mM KH2PO4: 10 ml/1 M solution
    6. Fill the column up to 800 ml with dH2O and add magnet bar
    7. Adjust with NaOH to pH 7.4 using a pH meter
    8. Filter sterilize with a 0.22 µm bottle-top filter under sterile conditions
    9. Use one filter for 1,000 ml buffer, filter 500 ml each in two sterile disposable bottles
    10. Store at 4 °C for up to four weeks (check before use, if cloudy prepare fresh) 
  3. Endothelial cell buffer (combine all components in a 1,000 ml cylinder)
    1. 153 mM NaCl: 30.6 ml/5 M solution
    2. 5.6 mM KCl: 5.6 ml/1 M solution
    3. 1.7 mM CaCl2: 1.7 ml/1 M solution
    4. 1.2 mM MgCl2: 1.2 ml/1 M solution
    5. 15 mM HEPES: 15 ml/1 M solution
    6. Fill the column up to 800 ml with dH2O and add magnet bar
    7. Weigh 10 g BSA and layer on top on the surface
    8. Place the cylinder at 4 °C (fridge or cold room) and cover with aluminum foil
    9. When BSA has completely dissolved (~30 min), put the column on a magnet stirrer
    10. Adjust with NaOH to pH 7.4 using a pH meter
    11. Filter sterilize with a 0.22 µm bottle-top filter under sterile conditions
    12. Use one filter for 1,000 ml buffer, filter 500 ml each in two sterile disposable bottles
    13. Store at 4 °C for up to four weeks (check before use, if cloudy prepare fresh)
  4. Collagenase II solution
    1. Prepare 2,400 U/ml collagenase II endothelial cell buffer
    2. Filter sterilize with a 0.22 µm bottle-top filter under sterile conditions
    3. Aliquot 2 ml into 15 ml tubes and store at -20 °C up to a year
  5. 25% BSA
    1. Fill a 2,000 ml Erlenmeyer flask with 400 ml PBS for BSA
    2. Weigh 100 g BSA and evenly layer on top on the surface and do not mix
    3. Place the flask in a -20 °C freezer overnight and cover with aluminum foil. The freezing process will allow the BSA to go into solution. We do not recommend using a magnetic stirrer for this step
    4. Thaw BSA in a 37 °C water bath. Put a weight ring on top of the flask to immerse completely in water. Swirl flask intermittently to facilitate the thawing process
    5. As soon as the BSA has completely thawed, add a magnet bar and let the solution stir until all residues stuck to the glass wall have been dissolved
    6. Place the funnel with the Whatman Grade 1573-1/2 filter paper in the 1,000 ml glass beaker and filter
    7. Under sterile conditions, filter BSA through a 0.45 µm bottle-top filter
    8. Aliquot (14 ml suggested) into 15 ml Falcon tubes and store at -20 °C up to a year
  6. Collagenase/Dispase solution
    1. Prepare a 100 mg/ml solution in molecular grade water
    2. Filter sterile into sterile 1.5 ml tube using a 1 ml syringe and the Millex-GV Filter 0.22 µm under sterile conditions
    3. Aliquot (45 µl suggested) into sterile 1.5 ml tubes and store at -20 °C up to a year
  7. DNase I solution
    1. Prepare a 1 mg/ml solution in molecular grade water. Do not vortex, mix gently
    2. Filter sterile into sterile 1.5 ml tube using a 1 ml syringe and the Millex-GV Filter 0.22 µm under sterile conditions
    3. Aliquot (5 µl suggested) into sterile 1.5 ml tubes and store at -20 °C up to a year
  8. FACS Buffer
    1. Prepare 10% FBS (endotoxin-low) in HBSS (50 ml)
    2. Store at 4 °C for up to two weeks (check for precipitate before use)
  9. Antibody dilutions (Table 1)

    Table 1. List of conjugated antibodies for flow cytometry

    *MECA-99 is a homemade antibody that was labeled with DyLightTM 488 Antibody Labeling Kit (Thermo Fisher Scientific). Antibody staining might vary from batch to batch. Determine optimal dilution for FACS if detected signals are too high or low.

Acknowledgments

This work was funded by the Edinger Institute, Frankfurt, Germany, the Stanford Institute for Immunity, Transplantation and Infection (ITI), Stanford, USA (C.J.C.), and by R01-GM37734 and R01-AI130471 from the National Institutes of Health (NIH) and an award from the Department of Veterans Affairs to E.C.B.
  We thank Stefan Liebner from the Edinger Institute, Frankfurt, Germany for providing the procedure of brain endothelial cell isolation for cultivation, which served as a starting protocol for this further developed and modified protocol.

Competing interests

The authors declare that there are no 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. and Friedman, A. (2012). Overview and introduction: the blood-brain barrier in health and disease. Epilepsia 53 Suppl 6: 1-6.
  2. 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.
  3. Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. and Greenleaf, W. J. (2013). Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 10(12): 1213-1218.
  4. Buenrostro, J. D., Wu, B., Chang, H. Y. and Greenleaf, W. J. (2015). ATAC-seq: A method for assaying chromatin accessibility genome-wide. Curr Protoc Mol Biol 109: 21.29.1-21.29.9.
  5. Czupalla, C. J., Liebner, S. and Devraj, K. (2014). In vitro models of the blood-brain barrier. Methods Mol Biol 1135: 415-437.
  6. 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.
  7. Fisher, J., Devraj, K., Ingram, J., Slagle-Webb, B., Madhankumar, A. B., Liu, X., Klinger, M., Simpson, I. A. and Connor, J. R. (2007). Ferritin: a novel mechanism for delivery of iron to the brain and other organs. Am J Physiol Cell Physiol 293(2): C641-C649.
  8. Haque, A., Engel, J., Teichmann, S. A. and Lonnberg, T. (2017). A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications. Genome Med 9(1): 75.
  9. Kruse, A., Hallmann, R. and Butcher, E. C. (1999). Specialized patterns of vascular differentiation antigens in the pregnant mouse uterus and the placenta. Biol Reprod 61(6): 1393-1401.
  10. Kukurba, K. R. and Montgomery, S. B. (2015). RNA sequencing and analysis. Cold Spring Harb Protoc 2015(11): 951-969.
  11. Lee, M., Kiefel, H., LaJevic, M. D., Macauley, M. S., Kawashima, H., O'Hara, E., Pan, J., Paulson, J. C. and Butcher, E. C. (2014). Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing. Nat Immunol 15(10): 982-995.
  12. Liebner, S., Corada, M., Bangsow, T., Babbage, J., Taddei, A., Czupalla, C. J., Reis, M., Felici, A., Wolburg, H., Fruttiger, M., Taketo, M. M., von Melchner, H., Plate, K. H., Gerhardt, H. and Dejana, E. (2008). Wnt/β-catenin signaling controls development of the blood-brain barrier. J Cell Biol 183(3): 409-417.
  13. Liebner, S., Czupalla, C. J. and Wolburg, H. (2011). Current concepts of blood-brain barrier development. Int J Dev Biol 55(4-5): 467-476.
  14. Liebner, S., Dijkhuizen, R. M., Reiss, Y., Plate, K. H., Agalliu, D. and Constantin, G. (2018). Functional morphology of the blood-brain barrier in health and disease. Acta Neuropathol 135(3): 311-336.
  15. Marchi, N., Granata, T., Ghosh, C. and Janigro, D. (2012). Blood-brain barrier dysfunction and epilepsy: pathophysiologic role and therapeutic approaches. Epilepsia 53(11): 1877-1886.
  16. Sealfon, S. C. and Chu, T. T. (2011). RNA and DNA microarrays. Methods Mol Biol 671: 3-34.
  17. Tung, J. W., Heydari, K., Tirouvanziam, R., Sahaf, B., Parks, D. R., Herzenberg, L. A. and Herzenberg, L. A. (2007). Modern flow cytometry: a practical approach. Clin Lab Med 27(3): 453-468, v.
  18. 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.
  19. Wolburg, H., Noell, S., Fallier-Becker, P., Mack, A. F. and Wolburg-Buchholz, K. (2012). The disturbed blood-brain barrier in human glioblastoma. Mol Aspects Med 33(5-6): 579-589.
  20. 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. (2018a). Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier. bioRxiv. Doi: 10.1101/242198.
  21. Yousef, H., Czupalla, C. J., Lee, D., Butcher, E. C. and Wyss-Coray, T. (2018b). Papain-based single cell isolation of primary murine brain endothelial cells using flow cytometry. Bio-protocol 8(22): e3091.
  22. Zlokovic, B. V. (2008). The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron 57(2): 178-201.

简介

脑内皮是一种高度专业化的血管结构,可维持中枢神经系统(CNS)的活动和完整性。以前的研究报道,大量神经病理学中脑内皮的完整性受到损害。因此,特别感兴趣的是建立一种方法,使研究人员能够研究和了解CNS内皮细胞的分子变化以及与小鼠疾病模型相关的潜在机制。过去,分离内皮细胞的方法涉及使用转基因报告小鼠或者由于纯度不足的细胞群和产量低而受到影响。

此协议基于完善的协议,这些协议经过修改和组合,允许使用荧光激活细胞分选(FACS)对高纯度脑内皮细胞群进行单细胞分离。简而言之,在仔细去除脑膜和解剖皮层/海马后,将脑组织机械均质化并在两个步骤中酶促消化,得到单细胞悬浮液。细胞用荧光染料缀合的抗体混合物染色,不仅识别脑内皮细胞,还可能污染细胞类型,如周细胞,星形胶质细胞和谱系细胞。使用流式细胞仪,将细胞群分离并直接分选到RNA裂解缓冲液中进行大量RNA分析(例如,RNA微阵列和RNA-Seq)或纯胎牛血清,以保持其他下游应用的可行性例如单细胞RNA-Seq和使用测序(ATAC-Seq)的转座酶 - 可接近染色质的测定。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,平均产量为来自四只成年小鼠的池的3×10 5个细胞。

【背景】 脑内皮充当通过血液循环的系统因素的界面。脑毛细血管内皮细胞构成血脑屏障(BBB),这是一种物理屏障,通过互连细胞的独特紧密连接蛋白形成来限制细胞旁通量,维持神经元环境的稳态,从而维持神经元功能(Liebner et al。 ,2011年和2018年)。 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不受控制的毒素,细菌,病毒和细胞交换(Abbott et al。,2010) 。为了完成这项任务,大脑内皮依赖于神经血管单元(NVU)内微调的微环境,其由内皮细胞和星形胶质细胞以及细胞外基质成分和小胶质细胞组成(Abbott和Friedman, 2012)。

BBB的功能和/或完整性在几种神经病理学中受到损害,例如阿尔茨海默病(AD),多发性硬化,癫痫和胶质母细胞瘤(Zlokovic,2008; Marchi et al。,2012; Wolburg et al。,2012)。这里描述的荧光激活细胞分选(FACS)单细胞分离方法已经在AD和炎症中脑内皮细胞的转录变化研究的背景下开发(未公开)。它是已公布的脑内皮细胞培养方案的修改(Liebner et al。,2008; Czupalla et al。,2014),旨在将脑EC分离为单细胞悬液,同时保留许多内皮表面抗原用于流式细胞术。我们已成功应用该方法协同研究老年系统性环境对海马神经发生和小胶质细胞活化的影响以及血管细胞粘附分子1(VCAM-1)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。 (Yousef et al。,2018a)。对于替代的脑内皮细胞分离方案 - 稍快一点,但利用较不温和的酶消化 - 参见Yousef 等(2018b)。

在过去的十年中,脑内皮细胞分离的几种方案主要用于BBB发育研究(Daneman et al。,2010; Vanlandewijck et al。,2018)。然而,这些技术依赖于转基因内皮细胞标记物的存在,因此可能不容易应用于转基因疾病小鼠模型。此外,取决于所用的消化酶,破坏了脑内皮细胞检测所必需的表位,例如CD31。该方案允许使用保留内皮特异性表位的温和酶促消化步骤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(和周细胞)。

关键字:CNS, 脑内皮细胞, 周细胞, 血脑屏障, 神经血管单元, 单细胞分离, RNA-Seq, RNA微矩阵

材料和试剂

  1. Falcon 15 ml锥形离心管(Fisher Scientific,Corning,目录号:352096)
  2. Falcon 50 ml锥形离心管(Fisher Scientific,Corning,目录号:352070)
  3. 瓶顶真空过滤器,孔径0.45μm(MilliporeSigma,Corning,目录号:CLS430514-12EA)&nbsp;
  4. 瓶顶真空过滤器,孔径0.22μm(MilliporeSigma,Corning,目录号:CLS430513-12EA)&nbsp;
  5. 一次性无菌瓶(Fisher Scientific,Corning,目录号:09-761-10)
  6. 1.5 ml Snap-Cap微量离心机Safe-Lock TM管(Eppendorf,目录号:022363204)
  7. 1毫升带滑盖的胰岛素注射器(BD,目录号:329654)
  8. 1毫升Millex-GV过滤器,0.22微米(MilliporeSigma,目录号:SLGV004SL)
  9. 培养皿100 x 21 mm(Thermo Fisher Scientific,Nunc TM,目录号:172931)
  10. 培养皿60 x 15 mm(Thermo Fisher Scientific,Nunc TM,目录号:150326)
  11. 血清移液管10 ml,无菌(SARSTEDT,目录号:86.1254.025)
  12. 可选:5毫升锥形管(Eppendorf,目录号:0030119487)(见注释)
  13. 血清移液器5 ml,无菌(SARSTEDT,目录号:86.1253.025)
  14. 带有细胞过滤器卡帽的5 ml试管(Falcon,目录号:352235)
  15. 一次性硼硅酸盐玻璃巴斯德吸管,高压灭菌(Fisher Scientific,目录号:13-678-20C)
  16. 螺帽微量离心管,1.5 ml(VWR,目录号:89004-290)
  17. 玻璃或塑料烧杯,1,000毫升和200毫升(实验室特定)
  18. 高压灭菌的Whatman TM定性滤纸,CF 12级(Sigma-Aldrich,目录号:WHA10535097)(见注释)
  19. Whatman TM 1573-1 / 2级定性滤纸(GE Healthcare,目录号:09-927-210)
  20. 一次性手术刀刀片,无菌(Integra TM Miltex®,目录号:4-123)
  21. 成年小鼠,6-12周,例如,C57 / Bl6(The Jackson Laboratory,目录号:000664)
  22. 去离子过滤水(dH 2 O)(Merck Millipore,Milli-Q)
  23. 牛血清白蛋白,组分V,热休克处理(BSA)(Fisher Scientific,Fisher BioReagents TM,目录号:BP1600-1)
  24. 氯化钠(NaCl)(MilliporeSigma,目录号:S3014)
  25. 氯化钾(KCl)(MilliporeSigma,目录号:PX1405)
  26. 氯化钙(CaCl 2)(MilliporeSigma目录号:102391)
  27. 氯化镁(MgCl 2)(MilliporeSigma目录号:M8266)
  28. HEPES(Thermo Fisher Scientific,Gibco TM,目录号:15630080)
  29. 氢氧化钠(NaOH),颗粒(MilliporeSigma目录号:S8045)
  30. 水,分子生物学试剂(MilliporeSigma,目录号:W4502-1L)
  31. 乙醇70%W / V(MilliporeSigma,目录号:EX0281-1)
  32. Red Blood Cell Lysis Buffer(MilliporeSigma,目录号:11814389001)
  33. 磷酸二氢钠(NaH 2 PO 4)(MilliporeSigma,目录号:S3139)
  34. 磷酸氢二钾(KH 2 PO 4)(MilliporeSigma,目录号:S3264)
  35. 胎牛血清(FBS),定义(HyClone TM,目录号:SH30070.03)
  36. 汉克斯平衡盐溶液(HBSS)(赛默飞世尔科技,Gibco TM,目录号:24020117)
  37. CD31(PECAM-1)单克隆抗体(390),PE-Cyanine7(eBioscience,目录号:25-0311-82)
  38. MECA-99(Eugene C. Butcher Laboratory)(Kruse et al。,1999; Lee et al。,2014)
  39. DyLight TM 488抗体标记试剂盒(赛默飞世尔科技,目录号:53024)
  40. Aminopeptidase N / CD13抗体(ER-BMDM1)(NOVUS Biologicals,目录编号:NB100-64843)
  41. ACSA-2(Miltenyi,目录号:130-097-674)
  42. 抗ACSA-2-PE,小鼠(Miltenyi,目录号:130-102-365)&nbsp;
  43. 抗小鼠CD45 PerCP-Cy5.5(eBioscience,目录号:45-0451-82)
  44. 抗小鼠CD11a / CD18(LFA-1)PerCP / Cy5.5(BioLegend,目录号:141008)
  45. 抗小鼠CD11b PerCP-Cyanine5.5(eBioscience,目录号:45-0112-80)
  46. 抗小鼠TER-119 PerCP-Cyanine5.5(eBioscience,目录号:45-5921-82)
  47. UltraComp eBeads TM(eBioscience,目录号:01-2222-42)
  48. 碘化丙啶溶液(PI)(MilliporeSigma,目录号:P4864)
  49. 可选:RNeasy Plus Micro Kit(50)(QIAGEN,目录号:74034)
  50. 可选:Array,Mouse Gene 1.0 ST ARRAY(Affymetrix,目录号:901169)
  51. II型胶原酶(Biochrom Kg,目录号:C2-22)
  52. 胶原酶/ Dispase(MilliporeSigma,目录号:11097113001)
  53. 脱氧核糖核酸酶I(CellSystems,目录号:LS006331)
  54. 库存解决方案(参见食谱)
  55. PBS for BSA(见食谱)
  56. 内皮细胞缓冲液(见食谱)
  57. 胶原酶II溶液(见食谱)
  58. 25%BSA(见食谱)
  59. 胶原酶/ Dispase解决方案(见食谱)
  60. DNase I解决方案(参见食谱)
  61. FACS缓冲液(见食谱)
  62. 抗体稀释液(见食谱)

设备

  1. 秤和称重供应(实验室特定)
  2. DURAN® Erlenmeyer烧瓶,2,000 ml(DURAN,目录号:21 216 63)
  3. -20°C冰柜(特定实验室)
  4. Precision GP 10 L通用水浴(Precision Scientific,目录号:TSGP10)
  5. 便携式移液器® XP移液器控制器(DRUMMOND,目录号:4-000-101)
  6. Thermo Scientific TM Nalgene TM聚丙烯粉末漏斗(Thermo Scientific TM,目录号:42520150)
  7. 气缸,气缸,Grad。 Cls B,1,000毫升和100毫升
  8. 磁力搅拌器(Thermo Fisher Scientific,目录号:90-691-18)
  9. Fisherbrand TM带可拆卸枢轴环的圆形搅拌棒(Thermo Fisher Scientific,适合测量柱的尺寸)
  10. Fisherbrand TM accumet TM AB15 Basic和BioBasic TM pH / mV /°C米或其他pH计
  11. 高压灭菌器(特定实验室)
  12. 二氧化碳室(实验室专用)
  13. 隔热容器,包括盖子,装满冰
  14. 手术剪刀,夏普(精细科学工具,目录号:14002-16)
  15. 精细剪刀,钝器(精细科学工具,目录号:14108-09)
  16. Semken Forceps,Straight(精细科学工具,目录号:11008-13)
  17. GSC Go Science疯狂不锈钢刮刀(Fisher Scientific,目录号:S50788A)
  18. Dumont#3 Forceps(精细科学工具,目录号:11293-00)
  19. Dumont#5-陶瓷涂层镊子(精细科学工具,目录号:11252-50)
  20. Scalpel Handle#4(精细科学工具,目录号:10004-13)
  21. 真空泵(实验室专用)
  22. 用于锥形管的离心机5810 R回转桶(Eppendorf,型号:5810 R)
  23. PIPETMAN Classic P2(吉尔森,产品目录号:F144801)&nbsp;
  24. PIPETMAN Classic P10(Gilson,产品目录号:F144802)&nbsp;
  25. PIPETMAN Classic P100(吉尔森,产品目录号:F123615)&nbsp;
  26. PIPETMAN Classic P200(吉尔森,产品目录号:F123601)&nbsp;
  27. PIPETMAN Classic P1000(吉尔森,产品目录号:F123602)
  28. 4°C冰箱(特定实验室)
  29. 尼康SMZ 745,立体显微镜(尼康,型号:SMZ 745)
  30. BD FACSAria TM II或III细胞分选仪(BD Biosciences)

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)
  3. 可选:GeneSpring GX(安捷伦科技,版本:14.8)
  4. 可选:Partek® Genomics Suite®(Partek Incorporated)

程序

注意:

  1. 图1概述了提供各个步骤概述的过程的工作流程。
  2. 该方案不需要无菌技术,但尽可能使用无菌设备和试剂,以尽量减少潜在的污染和促进细胞活力。
  3. 用70%(体积/体积)乙醇(玻璃烧杯)消毒解剖工具。使用前让工具干燥。
  4. 预冷的内皮细胞缓冲液(参见食谱)和冰上的6cm培养皿。
  5. 在37°C水浴中解冻25%牛血清白蛋白(BSA)(参见食谱),轻轻颠倒试管,搅拌均匀,并在解冻后立即放冰。
  6. 在使用前在37°C水浴中解冻并预热2,400 U / ml胶原酶II(参见食谱)20分钟。
  7. 将离心机冷却至4°C。
  8. 该方案的产量很大程度上取决于体积和管尺寸的正确选择。下面描述的方案设计用于从四个成年小鼠脑中分离细胞(参见注释)。对于额外的大脑/实验组,在过程中添加额外的样品管。&nbsp;


    图1.纯化从成年小鼠的皮质/海马中分离的鼠脑内皮细胞的方案概述有关详细信息,请参阅此工作流程图中所示的程序A-H。该图像包含Motifolio工具包中的图形元素。


  1. 脑组织收获
    1. 使用二氧化碳窒息随后颈椎脱位(遵守机构指南)牺牲成年小鼠。一次收获多达四个大脑。
    2. 用70%(体积/体积)乙醇喷洒头部/颈部区域,并使用锋利的手术剪刀从躯干上取下头部(图2)。&nbsp;
    3. 在室温下小心地从头骨解剖大脑。保持脑组织的完整性至关重要,因为切入或刺穿组织可能导致脑膜切除和皮质/海马解剖过程中的困难。使用钝剪刀沿左侧和右侧切开头骨(通过脊髓伸展进入),同时轻轻向外推动剪刀尖端(图2)。&nbsp;
    4. 小心地将头骨的上半部分抬起几毫米,然后在颅骨中间进行另一次切割,并用镊子将颅骨块剥离(图2)。&nbsp;
    5. 用小刮刀将大脑从头骨上移开(图2)。
    6. 将大脑置于冰上6厘米的培养皿中,加入冰冷的内皮细胞缓冲液覆盖(图2)。


      图2.脑组织收获。程序A(成年野生型Balb / c小鼠)中描述的脑组织收获程序的说明。 (步骤1是动物牺牲)。

    注意:&nbsp;
    1. 从此开始,一次只处理一个大脑,直到另有说明为止。&nbsp;
    2. 确保本节所述的解剖过程每个大脑的处理时间不超过15分钟。在实施该技术的同时,在冰上的步骤中冷却覆盖在内皮细胞缓冲液中的组织以保持细胞活力。或者,如果显微镜设置允许,可以在冰上的培养皿中进行组织解剖。但是,在黑暗的背景下,脑膜更容易识别。

  2. 皮质/海马解剖
    1. 将大脑转移到空的10厘米培养皿中,用手术刀取出小脑(包括脑桥和髓质,如果适用)和嗅球,同时用镊子小心地稳定大脑。
    2. 将大脑置于高压灭菌的Whatman滤纸上,用无菌刮刀在表面轻轻滚动,粗略去除脑周围的脑膜层(图3)。由于脑组织柔软而粘稠,因此在滚动过程中应使用小刮刀小心地将其与滤纸分开,以尽量减少组织损失。皮质/海马微血管具有不同于脑膜血管的表型特征,并且后来的结果包括不希望的内皮细胞群的污染。
    3. 使用小刮刀(不是镊子)将组织转移到10厘米培养皿上,并用冰冷的内皮细胞缓冲液覆盖。
    4. 将培养皿置于双目显微镜下,继续使用陶瓷涂层Dumont#5镊子手动移除剩余的脑膜,同时用另一组镊子(Dumont#3或#5)稳定组织(图3)。尽管脑膜可能很难看到,因为它们很脆弱并且大部分是透明的,因此旨在去除所有的脑膜结构。特别注意“红色”组织区域,因为它们表明红细胞填充的血管,其在整个脑膜层形成网络,因此用作指示哪些区域必须从脑膜清除。
    5. 使用无菌手术刀刀片沿着半球间裂隙用干净的切口分开半球(图3)。
    6. 将一个半球转移到带有内皮细胞缓冲液的培养皿中,并继续解剖剩余一半的皮层和海马。
    7. 抓住一小块“白色”脑组织 - 先前由皮质包围,包括中脑,丘脑,下丘脑,基底前脑,尾状壳核,腹侧纹状体,前嗅核,以及此处未列出的其他脑区 - 在后区杜蒙钳#3或#5。然后以与工作台顶部几乎平行的角度刺入下面的组织,使用Dumont镊子#5像剪刀一样切开(图3)。继续沿着略微更“粉红色”的皮层切割,直到去除不需要的“发白”组织。理想情况下,您可以在解剖后看到皮层的边缘表面和嵌入的海马体(图3)。但是,如果不是这种情况,请继续逐个去除不需要的组织部件。&nbsp;
    8. 继续移除内侧纵裂隙中的脑膜并翻转半球,以彻底检查和清理皮质的另一侧。
    9. 将皮质/海马体转移到充满内皮细胞缓冲液的冰上的干净培养皿中。
    10. 对于第二个半球,通过步骤B6到B9,然后从步骤B1开始,使用下一个脑,直到所有大脑都被处理完毕(对于2 x 4个大脑,总共需要2小时;对于这部分程序,不要超过3小时,因为细胞活力会受到影响)。
    11. 此后,处理合并的组织。


      图3.程序B中描述的皮质/海马解剖的选定步骤。右下图显示了由绿色虚线表示的海马体的位置。&nbsp;

  3. 组织均质化和初级消化&nbsp;
    1. 通过重复添加内皮细胞缓冲液,旋转培养皿,然后使用真空泵小心抽吸,洗涤汇集的脑组织三次。
    2. 在最后一次抽吸后,使用两个无菌手术刀将组织切成小块,直到形成均匀的乳液。
    3. 将4ml内皮细胞缓冲液加入培养皿中,用10ml移液管上下吸移重悬组织。&nbsp;
    4. 将细胞悬浮液转移到保持在冰上的15ml Falcon管中。
    5. 重复步骤C3和C4两次以冲洗培养皿并用内皮细胞缓冲液加至15ml。
    6. 将细胞悬浮液在4℃下以300μL离心7分钟,并使用真空泵吸出上清液。&nbsp;
    7. 估计组织沉淀的体积(通常约1.5 ml),并以1:1:1的比例添加内皮细胞缓冲液以及预热的胶原酶II。用5毫升移液管轻轻上下移液。
    8. 将酶混合物在37℃水浴中孵育50分钟,并在孵育25和50分钟后彻底摇动管以均化悬浮液直至看不到白色团块。
    9. 通过将内皮细胞缓冲液加入15ml中止酶促消化(~4.5ml体积),并通过上下彻底吸移混合悬浮液。
    10. 将细胞悬浮液在4℃下以300μL离心7分钟,并使用真空泵吸出上清液。&nbsp;

  4. 髓鞘去除和红细胞耗尽
    1. 加入3毫升25%BSA并转移到新的15毫升管中。用3 ml 25%BSA再次冲洗原始管,然后加热至15 ml,将悬浮液与10 ml移液管充分混合。
    2. 在4℃以1,000μL离心30分钟以分离髓磷脂(顶部)并富集毛细管碎片(底部)(图1D)。
    3. 用真空泵吸出髓鞘层。在去除透明BSA上清液之前,切换到新的尖端以最小化细胞沉淀中的残留髓鞘。
    4. 为了消耗红细胞,将沉淀物在2ml红细胞裂解缓冲液中在室温下孵育90秒,偶尔摇动。用P1000移液管加入1 ml红细胞裂解缓冲液,将悬浮液转移到新的15 ml Falcon试管中,再用红细胞裂解缓冲液1 ml冲洗试管并混合。&nbsp;
    5. 通过加入13ml内皮细胞缓冲液抑制细胞裂解并将样品放回冰上。
    6. 将细胞悬浮液在4℃下以300μL离心7分钟,并使用真空泵吸出上清液,并在管中留下~2ml。用1 ml移液器小心地去除剩余的上清液。

  5. 二次消化 - 单细胞悬液
    1. 将细胞沉淀重悬于2ml内皮细胞缓冲液中,并将细胞悬浮液转移至新的15ml Falcon管中。
    2. 为了将微血管片段消化成单细胞悬浮液,加入1mg / ml胶原酶/ Dispase并将混合物在37℃水浴中孵育13分钟。
    3. 注意由DNA聚集的内皮微血管片段聚集体的形成。加入1μg/ ml DNase I,上下移液几次,直到微量容器用P1000移液管解离,并在37°C水浴中再孵育2分钟。&nbsp;
    4. 为了淬灭消化反应,加入13ml内皮细胞缓冲液并轻轻倒转管混合(不要上下移液)。
    5. 将离心细胞悬浮液在4℃下在300μLg/ g下离心10分钟。
    6. 使用真空泵吸出上清液,在管中留下约2ml。使用1 ml移液器小心地移除剩余的上清液并将细胞沉淀物储存在冰上。
    7. 将沉淀的细胞重悬于FACS缓冲液中(参见配方)。每个抗体混合鸡尾酒FACS样品的总重悬浮体积应为200μl,对于未染色的对照样品应为50μl(对于其他对照,请参见下面的注释部分)。&nbsp;
    8. 将重悬的细胞分配到适当标记的1.5ml管中,用于抗体混合物FACS样品(200μl)和未染色的对照(50μl)并储存在冰上。
    注意:&nbsp;
    1. FACS参数设置的重要控制包括单色正控制,以补偿通道溢出。由于样品池数量是一个限制因素,我们建议将补偿珠与未染色的细胞样本控制结合使用,以设置前向和侧向散射。
    2. 我们进一步建议使用FMO(荧光减1)对照建立适当的门控,其中细胞用除一个之外的所有抗体染色。这对于初始FACS设置尤其重要,并且在使用新抗体批次时应该重复,因为信号强度可能因批次而异。有关详细信息,请参阅Tung 等。 (2007)。&nbsp;
    3. 计划运行实验,仅用于在FACS设备上建立这些设置。在我们的手中,该程序具有高度可重复性,保存的实验设置可以通过微小的调整进行后续实验,最大限度地利用可用的细胞样本进行分选。&nbsp;

  6. FACS的免疫染色
    1. 标记用于实验中的每种荧光染料的5ml FACS试管,加上一种用于未染色的对照,并每管加入150μlFACS缓冲液(补偿对照)。
    2. 通过剧烈颠倒至少10次混合UltraComp eBeads并将三滴加入1.5ml管中并加入100μlFACS缓冲液。
    3. 将50μl稀释的补偿珠稀释液应用于每个补偿对照管。
    4. 添加用于FACS样品染色的相同稀释度(参见配方)的抗体(为PerCP-Cy5.5选择一种抗体)。
    5. 通过轻弹管充分混合,在黑暗中在冰上孵育20分钟。
    6. 将所有抗体添加到抗体混合物FACS样品中(参见食谱),并使用P100移液管(避免气泡)上下移液小心混合。
    7. 在黑暗中在冰上孵育30分钟。
    8. 在孵育期间,准备用于下游应用的收集管:
      1. RNA提取:制备两个1.5 ml螺旋盖管,每个含750μlRLTPlus裂解缓冲液(参见制造商指南)。确保体积不会显着低于750μl,因为低于裂解缓冲液的1:1的比例,分选的样品可能导致RNA质量降低。将收集管保持在室温下。
      2. 完整细胞:准备两个1.5 ml螺旋盖管,每个500μlFBS,并在冰上冷却。
    9. 通过向每个管中加入1ml FACS缓冲液(FACS样品以及补偿珠)洗掉多余的抗体,并在4℃下以300 x g 离心5分钟。
    10. 倾倒补偿珠管的上清液。
    11. 使用P1000然后用P200移液器小心地吸出FACS样品上清液。&nbsp;
    12. 将FACS样品沉淀重悬于300μl中,并将补偿珠重悬于200μlFACS缓冲液中。
    13. 在冰上和黑暗中保持补偿控制直到FACS。
    14. 向保持在冰上的未染色细胞样品中加入150μlFACS缓冲液,并如上所述充分混合。&nbsp;
    15. 在将FACS样品转移到带有细胞过滤器的5 ml试管的蓝色过滤器盖上之前,确保使用P200移液器分离潜在的样品细胞聚集体。
    16. 将细胞通过过滤器在4℃下以300 x g 旋转2分钟,并将样品在黑暗中储存在冰上。
    17. 将样品和1:10碘化丙啶(PI)预稀释(在FACS缓冲液中)转运至FACS设施。始终将细胞保持在冰上和黑暗中。
    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。
    5. 10%-25%的事件是CD31 / MECA-99 + 脑内皮细胞;分选的脑内皮细胞计数范围为1-5 x 10 5 细胞;四只成年小鼠的平均计数:3×10 5 细胞。

  7. 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4)。
    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,将1:3,000(1:300从1:10预稀释;等于0.33μg/ ml)PI加入活/死染色,因为它影响细胞活力。
    4. 对新批次的抗体运行FMO对照,以确定阳性细胞群并相应地设置门(图4)。有关更多详细信息,请参阅Tung et al。(2007)。


      图4.用于纯化单细胞内皮细胞的FACS门控策略。 :一种。细胞在向前门控(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的髓鞘。 B. FSC-A和FSC-W绘图用于区分单细胞与细胞双联体/聚集体。 C. PI摄取表示排除死细胞。 D.门控CD45,CD11a / b和Ter-119阴性细胞以排除红细胞,单核细胞/巨噬细胞和小胶质细胞。 E.应用CD13和ACSA-2染色以分别排除周细胞(橙色)和星形胶质细胞(红色)。 CD13阳性群体由周细胞和周细胞/内皮细胞双联体(橙色)组成,并且可以在内皮标志物CD31 / MECA-99(未显示)上进一步门控,导致双阴性群体代表周细胞。 F.CD31 / MECA-99双阳性细胞定义为脑内皮细胞群。蓝色数字表示父母门的事件百分比。点图显示了50k重新编码事件。

    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(图4A)。
      2. 绘制针对FSC-W的FSC-A以区分单细胞与细胞双联体/聚集体(图4B)。有关更多详细信息,请参阅Tung 等人(2007)。
      3. 排除PI阳性(死亡)细胞(图4C)。
      4. 通过门控CD45,CD11a / b和Ter-119阴性细胞排除红细胞,单核细胞/巨噬细胞和小胶质细胞(图4D)。
      5. 通过对双阴性群体进行门控排除周细胞(CD13 +)和星形胶质细胞(ACSA-2 +)(图4E)(见注释)。
      6. 在CD31 / MECA-99双阳性细胞上接门并从该门收集细胞以获得纯脑内皮细胞群(图4F)。记录100万个事件。
    6. 将细胞收集在含有(a)RLT Plus裂解缓冲液的1.5ml螺旋盖微量离心管中,用于直接细胞裂解和大量RNA下游应用或(b)FBS,用于需要完整/单细胞的下游应用。&nbsp;

  8. 选择可选的下游应用
    1. RNA微阵列(Sealfon和Chu,2011)(图5)。
    2. RNA-Seq,散装(Kukurba和Montgomery,2015)。
    3. 单细胞RNA-Seq(Haque et al。,2017)。
    4. 使用测序(ATAC-Seq)测定转座酶可及的染色质(Buenrostro 等人,2013和2015)。
    5. 我们不建议培养分选的内皮细胞,因为该过程会对影响表达谱的细胞产生应激。细胞可能附着在细胞外基质涂层表面,但不会充分扩散。


      图5.来自小鼠脑的FACS分选细胞群的质量控制。 A-d。分离微血管(MVT)(Fisher et al。,2007),并在纤连蛋白包被的载玻片上接种,用于脑内皮标记物CD31,周细胞标记物Desmin和星形胶质细胞标记物GFAP和Aqp4的双重免疫组织化学染色。表明使用机械组织解离的细胞分离不充分。黑色标度50微米,白色标度25微米。 MVT用作RNA微阵列研究的混合细胞群控制(E-G)。 E.使用流式细胞术分离的脑毛细血管内皮细胞(CAP),周细胞(PC),星形胶质细胞(AC)和小胶质细胞(MG)单细胞群的转录组的主成分分析(PCA)。为了纯化CAP和PC群体,应用概述的方案,而使用未公开的方法分离AC和MG。计算归一化EV的PCA,任何样品对之间的差异至少为两倍( P <0.05 [单向ANOVA]),原始表达(EV)> 140在至少一个样本群体的100%重复中。括号中的数字表示为每个主成分(PC)计算的总变异性的比例。每个数据点代表从2-8只成年C57 / Bl6小鼠汇集的组织中分选的细胞的生物学重复。分析表明明确定义的细胞群基于它们的细胞类型特异性转录组聚集在一起。 F.通过基于log 2归一化EV的样本MVT,CAP,PC,AC和MG与E中定义的基因列表的相关性进行分层聚类。每个末端分支代表来自如E.E.5中所述的分选细胞的独立生物学重复的单个微阵列分析的结果。通过选择的细胞类型定义基因标记的相关性的分层聚类:Aqp4,GFAP和Acsa-2(AC); Pdgfrb,CD13和Desmin(PC); CD45,CD11b,Iba-1和CD68(MG); Tie2,Podxl,Cldn5和CD31(CAP)。单个细胞类型聚集在一起并表达高水平的已知细胞类型特异性基因,表明高纯度细胞群。作为对照,MVT阵列显示存在周细胞,内皮细胞,MG和星形胶质细胞基因。&nbsp;

数据分析

下面描述的数据分析是指使用图5中所示的RNA微阵列技术进行的纯化质量控制转录组分析。对于脑内皮细胞和周细胞群的纯化,应用概述的方案,而使用不同的技术分离微血管,星形胶质细胞和小胶质细胞(Fisher) et al。,2007和Fisher et al。,未发表)。

  1. RNA样品的质量和数量通过斯坦福大学PAN工厂的Agilent Bioanalyzer Total RNA Pico Euk芯片读数确定。
  2. 使用表达分析(Q2Solutions)进行扩增,标记和杂交,将3至50纳克的总RNA(最小RNA完整性数(RIN)为8)用于扩增,标记和杂交。将样品在Affymetrix Genechip Mouse Gene 1.0 ST阵列上杂交。&nbsp;
  3. 应用GeneSpring GX样品预处理和默认标准化(RMA-16)。所有RNA样品的动态范围至少为40。
  4. GeneSpring GX和Partek Genomic Suite软件用于处理和分析转录组数据。计算归一化EV的主成分分析(PCA),任何样品对之间的差异至少为2倍( P <0.05 [单向ANOVA],渗透100倍多重测试, Benjamini-Hochberg校正)并且在至少一个样本群体的100%重复中具有> 140的原始表达(EV)。&nbsp;
  5. 每个PCA数据点代表从2-8只成年C57 / B16小鼠(年龄:6-14周[细胞]和4个月[MVT])汇集的组织中分选的细胞的生物学重复。
  6. 通过基于log 2标准化EV(图5F)将所有样品与PCA分析中定义的基因列表相关联来进行分层聚类。每个末端分支代表来自分选细胞的独立生物学重复的单个微阵列分析的结果。
  7. 通过选择的细胞类型定义基因标记:Aqp4,GFAP和Acsa-2(星形胶质细胞)的相关性进行分层聚类; Pdgfrb,CD13和Desmin(周细胞); CD45,CD11b,Iba-1和CD68(小胶质细胞); Tie2,Podxl,Cldn5和CD31(脑毛细血管内皮细胞)。&nbsp;

笔记

  1. 该方案也可以应用于来自年龄较大的成年小鼠的脑组织。请注意,产量可能随着年龄的增长而下降,或者可能受转基因表达的影响。
  2. 该协议也可以应用于个体大脑。如果适用,使用5 ml而不是15 ml管和1.5 ml锥形管,按比例缩小缓冲液和酶溶液体积(例如,对于一个脑,使用指示体积的四分之一;孵育时间仍然是相同)。
  3. 将Whatman滤纸切成合适的尺寸(例如,18 x 12 cm),并用铝箔或可高压灭菌的容器包裹高压灭菌器。
  4. 确保在移除脑膜和组织解剖过程中脑组织保持湿润状态。
  5. 由于脂肪含量高,脑组织颗粒相当不稳定且粘稠。始终使用锥形管,并确保使用摇摆式离心机收集管底部的细胞。不要滗出上清液,而是使用真空泵进行大量吸入,并使用手持移液器进行小体积吸取。
  6. 抗体浓度是建议,应该进行测试,因为染色强度可能因批次而异。
  7. CD13 +周细胞群由单个周细胞以及周细胞/内皮细胞双联体组成。 CD13 +细胞可以在两种内皮标记物上进一步门控(未显示)。得到的CD31 / MECA-99双阴性群体代表周细胞。值得注意的是,纯周细胞的产量相当低(1-1.5×10 4)并且需要更多的起始材料,通常多达8只小鼠。
  8. 产生MECA-99抗体并在内部与DL488缀合。如果您希望通过其他颜色延伸荧光染料面板,可以选择不同的共轭。 Eg ,在我们的一项研究中,我们在体内注射VCAM-1-DL488 并用MECA-99-DL405染色细胞(Yousef 等。,2018a)。
  9. RNA样本不应放在冰上,而是在分拣完成后立即处理。或者,将RNA样品置于-80°C的RLP Plus缓冲液中进行短期储存(最多四周)。

食谱

  1. 储备溶液(在dH 2 O)
    1. 2 M NaOH(50 ml)
    2. 5 M NaCl(100 ml)
    3. 1 M KCl(50 ml)
    4. 1 M CaCl 2(50 ml)
    5. 1 M MgCl 2(50 ml)
    6. 1 M Na 2 HPO 4(50 ml)
    7. 1 M KH 2 PO 4(50 ml)
    注意:&nbsp;
    1. 储备溶液b-g用于制备内皮细胞缓冲液,PBS用于BSA溶液。
    2. 在50 ml Falcon试管中分装并在室温下储存(使用前检查沉淀物,并根据需要准备新鲜的。)
  2. 用于BSA的PBS(将所有组分组合在1,000毫升的圆筒中)
    1. 140mM NaCl:28.4ml / 5M溶液
    2. 0.2mM CaCl 2,:0.2ml / 1M溶液
    3. 0.2mM MgCl 2:0.2ml / 1M溶液
    4. 10mM Na 2 HPO 4:10ml / 1M溶液
    5. 10mM KH 2 PO 4:10ml / 1M溶液
    6. 用dH 2 O填充柱子至800毫升并加入磁棒
    7. 使用pH计将NaOH调节至pH 7.4
    8. 在无菌条件下用0.22μm瓶顶过滤器过滤灭菌
    9. 使用一个过滤器加入1,000毫升缓冲液,每个过滤500毫升无菌一次性瓶子
    10. 在4°C下储存长达四周(使用前检查,如果阴天准备新鲜)&nbsp;
  3. 内皮细胞缓冲液(将所有成分合并在1000毫升的圆筒中)
    1. 153mM NaCl:30.6ml / 5M溶液
    2. 5.6mM KCl:5.6ml / 1M溶液
    3. 1.7mM CaCl 2,:1.7ml / 1M溶液
    4. 1.2mM MgCl 2:1.2ml / 1M溶液
    5. 15mM HEPES:15ml / 1M溶液
    6. 用dH 2 O填充柱子至800毫升并加入磁棒
    7. 称重10克BSA并在表面上铺设
    8. 将圆筒放在4°C(冰箱或冷藏室)并用铝箔覆盖
    9. 当BSA完全溶解(约30分钟)时,将柱子放在磁力搅拌器上
    10. 使用pH计将NaOH调节至pH 7.4
    11. 在无菌条件下用0.22μm瓶顶过滤器过滤灭菌
    12. 使用一个过滤器加入1,000毫升缓冲液,每个过滤500毫升无菌一次性瓶子
    13. 在4°C下储存长达四周(使用前检查,如果混浊准备新鲜)
  4. 胶原酶II溶液
    1. 准备2,400 U / ml胶原酶II内皮细胞缓冲液
    2. 在无菌条件下用0.22μm瓶顶过滤器过滤灭菌
    3. 将2毫升分装到15毫升管中,在-20°C下储存长达一年
  5. 25%BSA
    1. 用含有400ml PBS的2,000ml Erlenmeyer烧瓶填充BSA
    2. 称取100 g BSA,均匀涂在表面上,不要混合
    3. 将烧瓶置于-20°C冰箱中过夜,并用铝箔覆盖。冻结过程将使BSA进入解决方案。我们不建议在此步骤中使用磁力搅拌器
    4. 在37°C水浴中解冻BSA。将重量环放在烧瓶顶部,完全浸入水中。间歇地旋流烧瓶以促进解冻过程
    5. 一旦BSA完全解冻,加入磁棒,搅拌溶液直至粘在玻璃壁上的所有残留物都溶解
    6. 将带有Whatman Grade 1573-1 / 2滤纸的漏斗放入1,000 ml玻璃烧杯中并过滤
    7. 在无菌条件下,通过0.45μm瓶盖过滤器过滤BSA
    8. 将等分试样(建议14毫升)加入15毫升Falcon试管中,在-20°C下保存至一年
  6. 胶原酶/ Dispase解决方案
    1. 在分子级水中制备100 mg / ml溶液
    2. 在无菌条件下使用1ml注射器和0.22μmMillex-GV过滤器无菌过滤至无菌1.5ml管中
    3. 将等分试样(建议45μl)放入无菌的1.5ml管中,并在-20℃下储存长达一年
  7. DNase I解决方案
    1. 在分子级水中制备1 mg / ml溶液。不要涡旋,轻轻混合
    2. 在无菌条件下使用1ml注射器和0.22μmMillex-GV过滤器无菌过滤至无菌1.5ml管中
    3. 将等分试样(建议5μl)放入无菌的1.5ml试管中,并在-20℃下储存长达一年
  8. FACS缓冲区
    1. 在HBSS(50 ml)中制备10%FBS(内毒素低)
    2. 在4°C下储存长达两周(使用前检查沉淀物)
  9. 抗体稀释液(表1)

    表1.用于流式细胞术的缀合抗体列表

    * MECA-99是用DyLight TM 488抗体标记试剂盒(Thermo Fisher Scientific)标记的自制抗体。抗体染色可能因批次而异。如果检测到的信号太高或太低,确定FACS的最佳稀释度。

致谢

这项工作由德国法兰克福Edinger研究所,斯坦福免疫,移植和感染研究所(ITI),美国斯坦福(CJC)以及美国国立卫生研究院(NIH)的R01-GM37734和R01-AI130471资助。 )以及退伍军人事务部对欧洲央行的奖励
&NBSP;我们感谢来自德国法兰克福Edinger研究所的Stefan Liebner提供用于培养的脑内皮细胞分离程序,该程序作为该进一步开发和修改的方案的起始方案。

利益争夺

作者声明没有利益冲突。

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

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引用: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. DOI: 10.21769/BioProtoc.3092.
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