参见作者原研究论文

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Mar 2019
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FACS Enrichment of Total Interstitial Cells and Fibroblasts from Adult Mouse Ventricles
流式细胞术富集成年小鼠心室间质细胞和成纤维细胞   

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Abstract

Besides cardiomyocytes, the heart contains numerous interstitial cell types, including cardiac fibroblasts, endothelial cells, immune (myeloid and lymphoid) cells, and mural cells (pericytes and vascular smooth muscle cells), which play key roles in heart repair, regeneration, and disease. We recently published a comprehensive map of cardiac stromal cell heterogeneity and flux in healthy and infarcted hearts using single-cell RNA sequencing (scRNA-seq) (Farbehi et al., 2019). Here, we describe the FACS (Fluorescent Activated Cell Sorting)-based method used in that study for isolation of two cardiac cell fractions from adult mouse ventricles: the total interstitial cell population (TIP; non-cardiomyocytes) and enriched (Pdgfra-GFP+) cardiac fibroblasts.

Keywords: Adult mouse heart (成年小鼠的心脏), Cardiac interstitial cells (心脏间质细胞), Cardiac fibroblasts (心肌成纤维细胞), Collagenase digestion (胶原酶消化), Fluorescent Activated Cell Sorting (FACS) (荧光活化细胞分选)

Background

Cardiovascular disease, including myocardial infarction (MI), is the leading cause of death worldwide. As adult mammalian hearts have a very limited capacity to repair damaged myocardium following injury, considerable efforts have been made to augment endogenous repair processes and stimulate heart regeneration (Tzahor and Poss, 2017). However, the structural and cellular complexity of the heart presents challenges to obtaining a complete mechanistic understanding of its functional and repair processes.


Cardiomyocytes are the primary functional cell type in the heart; however, they coexist within a complex support network of interstitial cells, which include fibroblasts, vascular, immune, and nerve cells. Cardiac interstitial cells constitute 60%-70% of total hearth cells and have essential regulatory functions in tissue homeostasis and repair (Frangogiannis, 2019). Ischaemic injuries, such as MI, induce a complex cascade of molecular and cellular events to clear necrotic cardiomyocytes and replace the affected area with a fibrotic scar (Tallquist and Molkentin, 2017). Thus, characterizing the cellular heterogeneity of the heart and its flux during the post-injury stages of inflammation and repair is necessary to both advance insights into disease mechanisms and facilitate the discovery of novel therapeutic targets.


Recent advances in single-cell technologies have enabled the systematic investigation of cellular heterogeneity in a wide range of tissues, yielding fresh insights into regulatory mechanisms underpinning cell states in development and disease (Marioni and Arendt, 2017). A comprehensive understanding of cellular complexity in the heart using single-cell methods requires developing reliable and efficient protocols to isolate the heavily interconnected and heterogeneous cardiac cell types in a relatively intact and healthy state. Here, we present a mechanical and enzymatic digestion protocol that relies on FACS to generate large numbers of high-quality, viable cardiac interstitial cells, as well as enriched fibroblasts, using lineage tracing of GFP+ cells from the ventricles of Pdgfra+/GFP mice. For details on the use and execution of this protocol, please refer to Chong et al., 2011 and Farbehi et al., 2019.

Materials and Reagents

  1. DNA LoBind 1.5 ml tubes (Eppendorf, catalog number: 022431005)

  2. 60 mm cell culture dish (Corning, catalog number: CLS430166)

  3. 100 mm cell culture dish (Corning, catalog number: CLS430167)

  4. 50 ml and 15 ml Falcon conical centrifuge tubes (Thermo Fisher Scientific, catalog numbers: 14-959-53A; 14-432-22)

  5. 5 ml and 10 ml Stripette serological pipettes (Corning, catalog numbers: CLS4487 and CLS4488)

  6. Sterile plastic transfer pipettes (Thermo Scientific Samco, catalog number: SAM-336-1S)

  7. 10 μl, 200 μl and 1,000 μl ART pipette tips (Sigma-Aldrich, catalog numbers: A2598, A3098, and A3948)

  8. 5 ml round bottom FACS tubes (Corning, catalog number: 352052)

  9. LS columns (Miltenyi Biotec, catalog number: 130-042-401)

  10. Surgical scalpel blades No. 22 (Swann-Morton, catalog number: 0208)

  11. 0.2 μm syringe filters (Thermo Fisher Scientific, catalog number: NC9103939)

  12. 40 μm cell strainers (Corning, catalog number: CLS431750)

  13. BD ultra-fine insulin syringe (Becton Dickinson, catalog number: 326719)

  14. 10 ml and 50 ml BD syringes (Becton Dickinson, catalog numbers: 305482 and 309653)

  15. Cannula (blunt-end 22-G needle) (Becton Dickinson, catalog number: 305156)

  16. Silk suture (Ethicon, catalog number: 768G)

  17. 70 ml specimen Jar (Sarstedt, catalog number: SAR00002)

  18. Dead cell removal Kit (Miltenyi Biotec, catalog number: 130-090-101)

  19. C57BL/6J mice (The Jackson Laboratory, JAX: 000664)

  20. PdgfraGFP/+ mice (The Jackson Laboratory, MGI: 2663656)

  21. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A2153)

  22. 1× Phosphate Buffered Saline (PBS), pH 7.4 (Thermo Fisher Scientific, catalog number: 10010023)

  23. Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, catalog number: 10100147)

  24. Red blood cell (RBC) lysis buffer (Sigma-Aldrich, catalog number: R7757)

  25. Collagenase type II (Worthington Biochemical Corporation, catalog number: LS004177)

  26. DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) (Thermo Fisher Scientific, catalog number: D1306)

  27. APC-coupled anti-PDGFRA (CD140a) antibody (Thermo Fisher Scientific, catalog number: 17-1401-81)

  28. Heparin (Pfizer Australia Pty Ltd, catalog number: AUST R 49232)

  29. Ketamine (Provet (NSW) Pty Ltd)

  30. Xylazine (Provet (NSW) Pty Ltd)

  31. FACS buffer (see Recipes)

  32. Collagenase type II in PBS (see Recipes)

Equipment

  1. Timer (Thermo Fisher Scientific, catalog number: 06664251)

  2. Hemocytometer (NEUBAUER, catalog number: HL-8100204 I)

  3. Fine surgical tools:

    Surgical scissors, Sharp (Fine Science Tools, catalog number: 14002-14)

    Dumont Forceps, Micro-Blunted Tips (Fine Science Tools, catalog number: 11253-25)

    Spring Scissors, 8 mm Cutting Edge (Fine Science Tools, catalog number: 15024-10)

    Graefe Forceps (Fine Science Tools, catalog number: 11051-10)

    Extra Fine Graefe Forceps (Fine Science Tools, catalog number: 11150-10)

    Delicate Suture Tying Forceps (Fine Science Tools, catalog number: 11063-07)

  4. Leica DFC295 digital microscope

  5. Refrigerated centrifuge (Thermo Fisher Scientific, catalog number: 75004270)

  6. Refrigerated microcentrifuge (Thermo Fisher Scientific, catalog number: 75002402)

  7. FACS Aria II (5 lasers, 100 μm nozzle) (BD Bioscience, catalog number: SCR_018091)

  8. QuadroMACS separator (Miltenyi Biotec, catalog number: 130-090-976)

  9. Shaker water bath (Analytical Instruments, catalog number: 7746-22220)

Procedure

  1. Before you start

    1. Set all centrifuges to 4°C.

    2. Set the water bath to 37°C and adjust the water level to cover half of the specimen jar.

    3. Place the collagenase type II stock bottle in a desiccator at room temperate (RT) for at least 30 min before opening.

      Note: Collagenase II is supplied as a lyophilized powder and is very hygroscopic. The hygroscopicity significantly affects the enzymatic activity if the powder is exposed to moisture. Thus, desiccation of the stock bottle helps ensure the long-term storage of the collagenase II stock. The stock bottle should not be opened in a humid environment.

    4. Pre-warm the red cell lysis buffer to RT.


    Note: Perform all steps on ice unless otherwise indicated. A simplified workflow of this protocol is illustrated in Figure 1A-1J.


  2. Method

    1. Inject the PdgfraGFP/+ mouse with heparin (200 IU/mouse) to prevent microthrombus formation in the coronary circulation.

      Note: In the original eLife paper (Farbehi et al., 2019), experimental mice were sacrificed by cervical dislocation without heparin administration, and the heart cavity was flushed manually after excision. Here, we present a revised method in which mice are heparinized and the heart removed, allowing flushing of the coronary arteries by ex vivo retrograde perfusion to minimize contamination of blood cells. In this method, euthanasia occurs by exsanguination under anesthesia.

    2. Ten minutes after heparin injection, anesthetize the mouse with an intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (20 mg/kg body weight).

    3. After sedation, check for the lack of pain using the toe-pinch reflex before proceeding to the thoracotomy.

    4. Incise the skin along the mid-sternal axis using fine surgical scissors and expose the rib cage. Cut the rib cage and separate the ribs to expose the thoracic cavity. Using fine forceps and surgical scissors, free the heart and ascending aorta from connective tissue. Cut the aorta 2-3 mm from the aortic valve, freeing the heart from the thoracic cage (Figure 1A).

    5. Immediately place the heart in a 100 mm Petri dish containing cold PBS.

    6. Trim excess soft tissue from the aorta. Cannulate the aorta and secure with silk suture as previously documented in video form Li et al. (2014) (Figure 1B). Make an incision in the right atrium to permit coronary sinus effluent to flow freely.

    7. Attach a 10-ml syringe filled with PBS to the cannula, taking care not to introduce any air bubbles.

    8. Initiate perfusion by gently pressing the syringe plunger, maintaining a steady flow rate until the heart is cleared of blood (the fluid should be running clear).

      Note: Alternative cardiac perfusion methods, e.g., Langendorff perfusion or whole body in situ perfusion, extend the protocol time significantly and may cause additional ischemic stress.

    9. After perfusion, remove the cannula and transfer the heart to a new 100 mm Petri dish containing PBS. Remove atria, great vessels, and top one-quarter of ventricles to avoid contaminating the samples with atrioventricular mesenchymal tissue (Figure 1C).

    10. Transfer the heart to a 60 mm Petri dish and mince it with a scalpel to obtain ~1-3 mm3 pieces. Cover the tissue in a small volume of collagenase solution (~500 µl) to keep it moist during mincing.

      Note: This step should be performed within a 5 min timeframe. Small pieces of tissue (~1-3 mm3) should be visible (Figure 1D). Excessive mincing generates a cell slurry which will negatively affect cell viability.

    11. Add 5 ml of collagenase solution to the dish and transfer the minced tissue into a 70 ml specimen jar using a plastic transfer pipette. Mix by gently pipetting the sample up and down to break down the clumps.

    12. Incubate the specimen jar at 37°C in a water bath with shaking (60 rpm) for 10 min (Figure 1E). Break down clumps by intermittent pipetting while avoiding bubble formation.

    13. After 10 min of incubation, remove the sample from the water bath and let the pieces settle down to the bottom of the specimen jar. Transfer the supernatant (cell suspension) to a 50 ml Falcon tube by filtering through a 40 μm sterile cell strainer to remove cardiomyocytes and undigested debris (size of adult mouse cardiomyocytes ranges 100-200 μm) (Figure 1F).

    14. Add fresh 5 ml collagenase solution to the remaining undigested tissue in the specimen jar, mix by gently pipetting up and down, and repeat Steps B12-B13 further two times further. Pool the cell suspension from each heart in the same designated 50 ml Falcon tube. Centrifuge the cell suspension at 300 × g for 5 min and discard the supernatant.

      Note: At this stage, most of the tissue should be completely digested; however, if larger pieces are visible, the incubation period should be extended to ~15 min, and/or another round of digestion with 5 ml collagenase solution should be done.

    15. Resuspend the pellet in 1 ml of red cell lysis buffer and incubate at RT for 1 min (Figure 1G).

    16. Centrifuge the tube at 300 × g for 5 min and discard the supernatant.

    17. Gently resuspend the pellet in 1 ml of ice-cold FACS buffer and transfer the contents into 1.5 ml Eppendorf tubes. Remove the supernatant carefully by aspiration without disturbing the cell pellet.

    18. Centrifuge the tube at 300 × g for 5 min after washing, discard supernatant, and resuspend the pellet in 200 µl of dead cell removal microbeads; mix well and incubate for 15 min at RT (Figure 1H).

    19. During the incubation, prepare 15 ml (per heart) of 1× column binding buffer from the 20× stock solution (included in the dead cell removal kit), place MACS LS columns in the magnetic field of a QuadroMACS Separator, and precondition the column with 3 ml of 1× binding buffer (Figure 1I). Discard the flow-through.

    20. Load the cell suspension into the LS column and slowly add the remaining 12 ml of 1× binding buffer to each column, collecting the flow-through into a 15 ml tube placed on ice. The flow-through contains live cells.

    21. Centrifuge the tubes at 300 × g for 5 min. After discarding the supernatant, resuspend the pellet in 1 ml of ice-cold FACS buffer and transfer the contents into a 1.5 ml Eppendorf tube.

    22. Centrifuge at 300 × g for 5 min and resuspend the pellet in 500 µl of ice-cold FACS buffer.

    23. Transfer 100 µl of the cell suspension into a 5 ml FACS tube and dilute with ice-cold FACS buffer to achieve a cell density of approximately 1 million cells per ml. Add DAPI (final concentration: 100 ng/ml) to the cell suspension and proceed to FACS sorting (Figure 1J).



      Figure 1. Experimental workflow in graphical form


    24. Acquire cells on the BD FACS Aria II sorter fitted with a 100 μm nozzle (30 kHz, 20 PSI). Adjust the flow rate to achieve ~1,000 events/second. After establishing the gates (see below), sort TIP cells (Figure 1D) with 4-way purity precision mode in a 1.5 ml Eppendorf tube containing 50 μl FACS buffer. Once the desired number of TIP cells are sorted, sort GFP+ cells (Figure 1E).

      Gating strategy:

      1. Set FSC-A vs SSC-A gate to include TIP cells and exclude the remaining cardiomyocytes, debris, and cell aggregates (Figure 2A).

      2. Gate single cells and exclude cell aggregates based on FSC gating (Figure 2B).

      3. Gate single cells and exclude cell aggregates based on SSC gating (Figure 2C).

      4. Gate the DAPI- fraction to sort for “Live TIP” cells (Figure 2D).

      5. From the “Live TIP” fraction, gate the GFP+ fraction to sort cardiac fibroblasts (Figure 2E) (Chong et al., 2011).

      Notes:

      1. Both unstained and single-stained samples should be prepared from the same cell suspension obtained from wild-type (C57BL/6J) mouse hearts. This will adjust the compensation parameter settings to set background fluorescence levels and establish the gate for GFP+ cells.

      2. If PdgfraGFP/+ mice are not available, a cell suspension obtained from wild-type (C57BL/6J) mouse hearts (at Step B22) should be stained with a fluorophore-conjugated anti-PDGFRA antibody (e.g., APC-conjugated anti-PDGFRA (CD140a) antibody) to sort cardiac fibroblasts as the “APC” positive fraction from the “Live TIP” fraction. TIP cells can be sorted as explained above for PdgfraGFP/+ mice (Step B24 and Figures 2A-2D).



    Figure 2. FACS gating strategy. (A-C) A typical workflow of sequential gating for doublet exclusion is shown. Total events were gated in a scatter plot (A) and subjected to FSC-based (B) and SSC-based (C) single-cell gating. (D) Live TIP cells were then gated as the DAPI- fraction. (E) Example of gating for GFP+ cells isolated from PdgfraGFP/+ mice.

Recipes

  1. FACS buffer

    Prepare 2% FBS in PBS and filter through a 0.2 µm syringe filter.

  2. Collagenase type II in PBS (working concentration: 265 Unit/ml)

    Prepare fresh solution each time. Dissolve collagenase powder in PBS and filter through a 0.2 µm syringe filter. Prepare 15 ml of collagenase solution for each heart sample.

    Note:  The enzymatic activity per unit mass (Unit/mg) of the lyophilized powder and weigh as required (Unit/ml) considering working concentration and number of samples.

Acknowledgments

This work was supported by funding from the National Health and Medical Research Council (1118576, 573707, 1105271, 1074286), Leducq Foundation (13CVD01, 15CVD03), Stem Cells Australia (SR110001002), St. Vincent’s Clinic Foundation (100711), Victor Chang Cardiac Research Institute and UNSW Sydney.

Competing interests

The authors declare no conflict of interest.

Ethics

Animal experiments were performed following the guidelines and with the approval of the Garvan Institute of Medical Research/St. Vincent’s Animal Ethics Committee, research approvals 16/03 and 16/10 (Validity period: 2016-2019).

References

  1. Chong, J. J., Chandrakanthan, V., Xaymardan, M., Asli, N. S., Li, J., Ahmed, I., Heffernan, C., Menon, M. K., Scarlett, C. J., Rashidianfar, A., Biben, C., Zoellner, H., Colvin, E. K., Pimanda, J. E., Biankin, A. V., Zhou, B., Pu, W. T., Prall, O. W. and Harvey, R. P. (2011). Adult cardiac-resident MSC-like stem cells with a proepicardial origin. Cell Stem Cell 9(6): 527-540.
  2. Farbehi, N., Patrick, R., Dorison, A., Xaymardan, M., Janbandhu, V., Wystub-Lis, K., Ho, J. W., Nordon, R. E. and Harvey, R. P. (2019). Single-cell expression profiling reveals dynamic flux of cardiac stromal, vascular and immune cells in health and injury. Elife 8: e43882.
  3. Frangogiannis, N. G. (2019). Cardiac fibrosis: Cell biological mechanisms, molecular pathways and therapeutic opportunities. Mol Aspects Med 65: 70-99.
  4. Li, D., Wu, J., Bai, Y., Zhao, X. and Liu, L. (2014). Isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy. J Vis Exp(87): 51357.
  5. Marioni, J. C., and Arendt, D. (2017). How Single-Cell Genomics Is Changing Evolutionary and Developmental Biology. Annu Rev Cell Dev Biol 33: 537-553.
  6. Tallquist, M. D. and Molkentin, J. D. (2017). Redefining the identity of cardiac fibroblasts. Nat Rev Cardiol 14(8): 484-491.
  7. Tzahor, E. and Poss, K. D. (2017). Cardiac regeneration strategies: Staying young at heart. Science 356(6342): 1035-1039.

简介

[摘要]除心肌细胞外,心脏还包含多种间质细胞类型,包括心脏成纤维细胞,内皮细胞,免疫(髓样和淋巴样)细胞和壁细胞(周细胞和血管平滑肌细胞),它们在心脏修复,再生中起关键作用。,和疾病。最近,我们使用单公布心肌间质细胞的异质性和流量的全面的地图健康与心肌梗死后-细胞RNA测序(scRNA-SEQ )(Farbehi等,2019) 。在这里,我们描述了该研究中使用的基于FACS(荧光激活细胞分选)的方法,该方法用于从成年小鼠脑室中分离出两个心肌细胞级分:总的间质细胞群(TIP;非心肌细胞)和富集的(Pdgfra -GFP + )心脏成纤维细胞。



[背景]心血管疾病,包括心肌梗死(MI) ,是全球死亡的首要原因。作为成年哺乳动物心中都有一个容量非常有限损伤后修复受损心肌,已经取得了相当大的努力,以增加内源性修复过程ES和刺激心脏再生(Tzahor和POSS,2017年)。但是,心脏的结构和细胞复杂性对获得对其功能和修复过程的完整机械理解提出了挑战。

心肌细胞是心脏的主要功能细胞类型。然而,它们的间质细胞,包括成纤维细胞,血管,免疫的复杂的支持网络内共存,和神经细胞。心肌间质细胞构成60%- 70%的总细胞炉床和在组织稳态和修复必要的调节功能(Frangogiannis,2019) 。我schaemic injur IES ,如MI,诱导分子和细胞事件,以明确坏死心肌细胞的复杂级联,并与纤维化瘢痕更换受影响的区域(Tallquist和Molkentin,2017) 。因此,characteri ž期间炎症和修复损伤后阶段荷兰国际集团的心脏,它的通量的细胞异质性是必要的,以双方事先见解疾病机制,并促进了新的治疗靶点的发现。

在单细胞技术的最新进展已启用的细胞异质性在广泛的组织系统的调查,产生新的见解调节机制托底细胞状态的发育和疾病(MARIONI和阿伦特,2017) 。细胞的复杂性,使用单细胞方法的心脏全面了解需要开发荷兰国际集团可靠和有效的协议,在一个相对完整和健康状态,以隔离大量互连和异质性心肌细胞类型。在这里,我们介绍了一种机械和酶促消化方案,该方案依赖于FACS来生成大量高质量,可行的心脏间质细胞以及富集的成纤维细胞,并使用来自Pdgfra + / GFP小鼠心室的GFP +细胞的谱系追踪。有关此协议的使用和执行的详细信息,请参阅Chong等人,2011和Farbehi等人,2019。

关键字:成年小鼠的心脏, 心脏间质细胞, 心肌成纤维细胞, 胶原酶消化, 荧光活化细胞分选

材料和试剂
DNA LoBind 1.5 ml管(Eppendorf ,目录号:022431005 )
60 mm细胞培养皿(Corning,目录号:CLS430166)
100 mm细胞培养皿(Corning,目录号:CLS430167)
50 ml和15 ml F型圆锥锥形离心管(Thermo Fisher Scientific,目录号:14-959-53A; 14-432-22)
5 ml和10 ml Stripette血清移液管(Corning,目录号:CLS4487和CLS4488)
无菌塑料移液管(Thermo Scientific Samco ,目录号:SAM-336-1S)
10 μ升,200 μ升和1 ,000 μ升ART移液器吸头(Sigma-Aldrich公司,目录号:A2598,A3098 ,和A3948)
5 ml圆底FACS管(Corning,目录号:352052)
LS色谱柱(Miltenyi Biotec ,目录号:130-042-401)
22号手术刀刀片(Swann-Morton,目录号:0208)
0.2 μ米注射器过滤器(热Fisher Scientific公司,目录号:NC9103939)
40级μ米细胞过滤(Corning公司,目录号:CLS431750)
BD超细胰岛素注射器(Becton Dickinson,目录号:326719)
10 ml和50 ml l BD注射器(Becton Dickinson,目录号:305482和309653)
插管(22-G钝针)(Becton Dickinson,目录号:305156)
丝线缝合(Ethicon,目录号:768G)
70 ml标本罐(Sarstedt ,目录号:SAR00002)
死细胞去除试剂盒(Miltenyi Biotec ,目录号:130-090-101)
C57BL / 6J小鼠(Jackson Laboratory,JAX:000664)
Pdgfra GFP / +小鼠(The Jackson Laboratory,MGI:2663656)
牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
1 ×磷酸盐缓冲盐水(PBS),pH 7.4(Thermo Fisher Scientific,目录号:10010023)
胎牛血清(FBS)(Thermo Fisher Scientific,目录号10100147)
红细胞(RBC)裂解缓冲液(Sigma-Aldrich,目录号:R7757)
II型胶原酶(沃辛顿生物化学公司,目录号:LS004177)
DAPI(4',6-二mid基-2-苯基吲哚,二盐酸盐)(Thermo Fisher Scientific,目录号:D1306)
APC偶联的抗PDGFRA(CD140a)抗体(Thermo Fisher Scientific,目录号:17-1401-81)
肝素(辉瑞澳大利亚有限公司,目录号:AUST R 49232)
氯胺酮(Provet (NSW)Pty Ltd)
赛拉嗪(Provet (NSW)Pty Ltd)
FACS缓冲区(请参阅配方)
PBS中的II型胶原酶(请参阅食谱)


设备


计时器(Thermo Fisher Scientific,目录号06664251)
血细胞计数器(NEUBAUER,目录号:HL-8100204 I)
精细手术工具:
手术剪刀,夏普(精细科学工具,目录号:14002-14)


杜蒙钳,微钝针头(精细科学工具,目录号:11253-25)


弹簧剪刀,8 mm切削刃(精细科学工具,目录号:15024-10)


Graefe镊子(精细科学工具,目录号:11051-10)


超细Graefe镊子(Fine Science Tools,目录号:11150-10)


精致的缝合线钳(精细科学工具,目录号:11063-07)


徕卡DFC295数字显微镜
冷藏离心机(Thermo Fisher Scientific,目录号:75004270)
冷冻微量离心机(Thermo Fisher Scientific,目录号:75002402)
FACS咏叹调II(5个激光器,100 μ米喷嘴)(BD Bioscience公司,目录号:SCR_018091)
QuadroMACS分离器(Miltenyi Biotec ,目录号:130-090-976)
摇床水浴(分析仪器,目录号:7746-22220)


程序


在你开始之前
将所有离心机设置为4℃。
设置的水浴中以37 ℃,并调整水位到样品罐的盖半部。
打开之前,将II型胶原酶储备瓶放在室温下的干燥器中至少30分钟。
注意:胶原酶II以冻干粉形式提供,吸湿性极强。该吸湿性显著影响的酶的活性,如果将粉末受潮。因此,储备瓶的干燥有助于确保胶原酶II储备的长期储存。股票瓶不应在打开一个潮湿的环境。


将红细胞裂解缓冲液预热至室温。


ñ OTE :执行上冰的所有步骤,除非另有说明。该协议的简化工作流程如图1A-1J所示。


方法
向Pdgfra GFP / +小鼠注射肝素(200 IU /小鼠),以防止在冠状动脉循环中形成微血栓。
注意:在原始的eLife论文中(Farbehi et al。,2019),在不给予肝素的情况下通过颈脱位法处死实验小鼠,并在切除后手动冲洗心腔。在这里,我们提出一种改良的方法,其中将小鼠肝素化并取出心脏,以通过离体逆行灌注使冠状动脉潮红,以最大程度地减少血细胞的污染。在这种方法中,麻醉下通过放血发生安乐死。


十英里nutes肝素注射后,麻醉小鼠用的氯胺酮(100mg / kg的体重)和腹腔注射甲苯噻嗪(20毫克/千克体重)。
镇静后,在开始开胸手术之前,先用脚尖反射检查是否无疼痛感。
使用精细的手术剪刀沿胸骨中轴线切开皮肤,并暴露肋骨笼。切开肋骨并分开肋骨以暴露胸腔。用细镊子和手术剪刀将心脏和升主动脉从结缔组织中释放出来。切主动脉2 -从主动脉阀3毫米,从胸廓释放心脏的(图1A) 。
立即将心脏在100毫米P含有冷PBS ETRI菜。
修剪主动脉多余的软组织。ç有环主动脉和用丝线固定先前在视频形式记录李等人。(2014年)(图1B)。在右心房切开一个切口,使冠状窦流出物自由流动。
附加一个10 -毫升注射器填充有PBS到所述套管,注意不要引入任何气泡。
轻轻按压注射器活塞开始灌注,维持稳定的流量,直至心脏被清除的血液(流体应该运行明确)。
注:替代心脏灌注的方法,例如,的Langendorff灌注或原位灌注全身,显著延长协议时间,并且可以导致额外的缺血性应激。


灌注后,取出的插管和心脏转移到一个新百毫米P含有PBS ETRI菜。除去心房,大血管,和一个顶部-季度心室的,以避免污染与房室间充质组织(图1C)的样品。
的心脏转移到60毫米P ETRI皿中并剁碎它与一个手术刀以获得〜1-3毫米3片。用少量的胶原酶溶液(〜500 µl)覆盖组织,以使其在切碎时保持湿润。
注意:此步骤应在5分钟内执行。小块的组织(约1-3 mm 3 )应可见(图1D)。切碎过多会产生细胞浆,会对细胞活力产生负面影响。


5毫升添加的胶原酶溶液的培养皿中并用塑料移液管将切碎的组织转移到70毫升样品广口瓶中。向上和向下轻轻吸打样品以混合,以打破团块。
将样品罐在水浴中于37 °C摇动(60 rpm)孵育10分钟(图1E)。通过间歇移液来消除团块,同时避免形成气泡。
温育10分钟后,除去从样品的水浴中,并让件定下来到样品罐的底部。将上清转移(细胞悬浮液),以50毫升˚F通过通过40过滤爱尔康管微米无菌细胞过滤以除去心肌细胞和未消化的碎片(成年小鼠心肌细胞的尺寸范围100-200微米)(图1F)。
添加新鲜5毫升胶原酶溶液到剩余的未消化的组织试样中的罐,通过轻轻地上下吹打混匀,并重复小号TEPS乙-12-乙13分进一步两次进一步。将来自每个心脏的细胞悬液汇集在相同的50 ml F alcon管中。将细胞悬液以300 × g离心5分钟,并弃去上清液。
注意:在此阶段,大多数组织应被完全消化。但是,如果可以看到较大的碎片,则应将孵育时间延长至15分钟左右,并且/或者应该用5 ml胶原酶溶液进行另一轮消化。


将沉淀重悬于1 ml红细胞裂解缓冲液中,并在室温下孵育1分钟(图1G)。
将试管以300 × g离心5分钟,并弃去上清液。
轻轻地将沉淀重悬于1 ml冰冷的FACS缓冲液中,并将内容物转移到1.5 ml E ppendorf管中。小心吸除上清液,不要干扰细胞沉淀。
洗涤后将试管以300 × g离心5分钟,弃去上清液,并将沉淀重悬于200 µl去除死细胞的微珠中;充分混合并在室温下孵育15分钟(图1H)。
在温育期间,准备15毫升(每心脏)1 ×从柱结合缓冲液的20 ×储备液(包含在死细胞去除试剂盒),地方MACS LS在一个的磁场列QuadroMACS分离器,和前提列用3 ml的1 ×结合缓冲液(图1I)。丢弃流通物。
加载的细胞悬浮液中的LS柱和慢慢加入1个的剩余12毫升×结合缓冲液到每个列,收集荷兰国际集团的流通入15ml试管置于冰上。流通中包含活细胞。
将试管以300 × g离心5分钟。弃去上清液后,将沉淀重悬于1 ml冰冷的FACS缓冲液中,并将内容物转移至1.5 ml E ppendorf管中。
以300 × g离心5分钟,然后将沉淀重悬于500 µl冰冷的FACS缓冲液中。
转移100微升的细胞悬浮液中,以5ml的FACS管和稀用冰冷的FACS缓冲液,以实现一个的细胞密度约为每ml 1百万细胞。将DAPI(终浓度:100 ng / ml)添加到细胞悬液,并进行FACS分选(图1J)。




图1.图形形式的实验工作流


在装有100μm喷嘴(30 kHz,20 PSI)的BD FACS Aria II分选机上采集细胞。调节流速以达到〜1 ,000事件/秒。建立门(参见下文)后,以1的4向纯度模式对TIP单元(图1D)进行排序。5毫升Ë ppendorf管含有50微升FACS缓冲液。排序所需数目的TIP细胞后,对GFP +细胞进行分类(图1E)。
门控策略:


集FSC-A对SSC-A栅包括TIP细胞和排除的剩余的心肌细胞,碎片,和细胞聚集体(图2A)。
门控单细胞并排除基于FSC门控的细胞聚集体(图2B)。
门控单细胞并排除基于SSC门控的细胞聚集体(图2C)。
栅极的DAPI -分数排序为“实时TIP”细胞(图2D)。
从所述“活TIP”分数,栅极的GFP +级分,以排序心脏成纤维细胞(图2E) (冲等人,2011) 。
注意小号:


两个未染色和单染色的样品应从来制备相同的来自野生获得的细胞悬浮液-型(C57BL / 6J)小鼠心脏。这将调整的补偿参数设置来设置背景荧光水平,建立大门口GFP +细胞。
如果PDGFRA GFP / +小鼠是不可用的,从野生获得的细胞悬浮液-型(C57BL / 6J)小鼠心脏(在小号TEP B22 )应该被沾上一个荧光团-偶联的抗PDGFRA抗体(例如,APC缀合的抗-PDGFRA(CD140a)抗体)从“ Live TIP”部分中将心脏成纤维细胞分类为“ APC”阳性部分。TIP细胞可以按照上文对Pdgfra GFP / +小鼠的说明进行分类(S tep B 24和图s 2A- 2 D)。




图2 。FACS门控策略。(AC)显示了用于双峰排除的顺序门控的典型工作流程。事件总数中的散点图(A)进行门控,并进行基于FSC-(B)和基于SSC-(C)单-细胞门控。(d)活TIP细胞随后门控为DAPI -分数。(É )Ë选通GFP的xample +从分离的细胞PDGFRA GFP / +小鼠。


[R ecipes


1. FACS缓冲区     

制备在PBS中的2个%FBS通过和过滤器一个0.2微米的针筒式过滤器。


2. PBS中的II型胶原酶(工作浓度:265单位/毫升)     

每次准备新鲜的溶液。通过溶解在PBS中胶原酶粉末和滤波器一个0.2微米的针筒式过滤器。为每个心脏样本准备15毫升胶原酶溶液。


注意:注意冻干粉末的单位质量酶活性(单位/ mg),并根据需要的重量(单位/ ml)考虑工作浓度和样品数量。




致谢


这项工作是由美国国家卫生和医学研究理事会(1118576,573707,1105271,资金支持1074286 ),Leducq基金会(13CVD01,15CVD03),干细胞澳大利亚(SR110001002),圣文森特诊所基金会(100711),张任谦心脏研究所和悉尼西南大学。


利益争夺


作者宣称没有利益冲突。


伦理


动物实验是按照指南进行的,并得到了加尔文医学研究所/圣保罗大学的批准。文森特动物伦理委员会(Vincent's Animal Ethics Committee),研究批准书16/03和16/10(有效期:2016-2019年)。


参考


冲,JJ,Chandrakanthan ,五,Xaymardan ,M.,阿斯利,NS,李,J.,艾哈迈德,一,赫弗南,C.,梅农,MK,斯嘉丽,CJ,Rashidianfar ,A.,Biben ,C. ,Zoellner ,H.,Colvin,EK,Pimanda ,JE,Biankin ,AV,Zhou B.,Pu,WT,Pral ,OW和Harvey,RP(2011)。成人心源性MSC样干细胞,起源于心外膜。细胞干细胞9(6):527-540。
Farbehi ,N.,Patrick,R.,Dorison ,A.,Xaymardan ,M.,Janbandhu ,V.,Wystub -Lis,K.,Ho,JW,Nordon ,RE和Harvey,RP(2019)。单细胞表达谱揭示了健康和损伤中心脏基质,血管和免疫细胞的动态通量。Elife 8:e43882。
Frangogiannis ,N.G .(2019年)。心脏纤维化:细胞生物学机制,分子途径和治疗机会。Mol Aspects Med 65 :70-99。
Li D,Wu,J.,Bai Y,Zhao,X. and Liu,L.(2014)。成年小鼠心肌细胞的分离和培养,用于细胞信号传导和体外心脏肥大。Ĵ显示精通(87):51357。
Marioni JC和Arendt D.(2017年)。单细胞基因组学如何改变进化生物学和发育生物学。Annu Rev Cell Dev Biol 33:537-553。
医学博士塔尔奎斯特(Tallquist)和法医学博士莫尔肯汀(Molkentin)(2017)。重新定义心脏成纤维细胞的身份。Nat Rev Cardiol 14(8):484-491。
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Copyright Farbehi et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Farbehi, N., Janbandhu, V., Nordon, R. E. and Harvey, R. P. (2021). FACS Enrichment of Total Interstitial Cells and Fibroblasts from Adult Mouse Ventricles. Bio-protocol 11(10): e4028. DOI: 10.21769/BioProtoc.4028.
  2. Farbehi, N., Patrick, R., Dorison, A., Xaymardan, M., Janbandhu, V., Wystub-Lis, K., Ho, J. W., Nordon, R. E. and Harvey, R. P. (2019). Single-cell expression profiling reveals dynamic flux of cardiac stromal, vascular and immune cells in health and injury. Elife 8: e43882.
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