基于流式细胞术的心肌B细胞定量和分析
Summary
在这里，我们报告了一种使用流式细胞术根据心肌B淋巴细胞在血管内或内皮空间中的位置对心肌B淋巴细胞进行定量和分化的协议。
Abstract
越来越多的证据表明，B淋巴细胞在心肌生理学和心肌适应损伤的背景下起着重要作用。然而，文献报告了关于心肌B细胞患病率的对比数据。据报道，B细胞既是啮齿动物心脏中最普遍的免疫细胞之一，要么存在，但患病率明显低于骨髓细胞，或者相当罕见。同样，几组人描述急性缺血性心肌损伤后心肌B细胞数量增加，但一组报告受损心肌B细胞数量没有变化。实施一种共享的、可重复的方法来评估心肌B细胞的患病率对于协调不同研究小组的观察结果至关重要，从而促进B细胞心肌相互作用研究的进展。根据我们的经验，文献中报道的看似相反的观察结果可能源于这样一个事实，即鼠心肌B细胞大多在血管内并与微血管内皮相连。因此，从小鼠心脏中回收的B细胞数量对用于清洁器官的灌注条件和所使用的消化方法非常敏感。在这里，我们报告了一个优化的协议，该协议以特定的方式考虑了这两个关键变量。该协议能够对鼠心肌B细胞的数量进行可重复的，基于流式细胞术的分析，并允许研究人员区分血管外与血管内心肌B细胞。
Introduction
B淋巴细胞是高度特化的免疫细胞，在适应性和先天免疫反应中都起着重要作用1。B细胞有两个主要群体：较小的B1细胞群，主要在胚胎生命期间产生，以及成年后在骨髓中产生的B2细胞群占主导地位1。在骨髓中成熟后，B细胞迁移到原代和次级淋巴器官。从那里，它们在通过血管和淋巴管的淋巴器官之间不断循环2。B细胞在其表面表达特异性抗体，其作为受体发挥作用。当B细胞遇到与其受体结合的抗原时，可以触发激活信号。活化的B细胞要么迁移到发现抗原的组织，要么回到骨髓，在那里它们可以成熟为产生抗体的浆细胞3，4。
最近，人们认识到心脏含有大量的B细胞。对啮齿动物的研究表明，B细胞在胚胎发育早期定植在心脏5，并且心肌相关B细胞大多是血管内幼稚的B2细胞粘附在内皮6，7上，其中B1细胞的比例很小7。仍有许多不确定的领域，但现有数据表明，B细胞在幼稚的心脏和心肌适应损伤的背景下都起着重要作用。
对幼稚鼠心脏的研究表明，在基线时心肌B细胞大多位于血管内间隙，粘附在内皮上（发现>95%的小鼠心脏B细胞位于血管内间隙）。发现这些B细胞的基因表达模式与从外周血分离的循环B细胞的基因表达模式不同。对B细胞缺陷动物的幼稚心脏和同系对照的分析发现，缺乏B细胞的动物心脏较小，射血分数较高6。所有这些证据表明，B细胞可能调节心肌生长和/或心肌功能，不仅间质，而且血管内B细胞都可能负责这种观察。还发现B细胞调节心肌常驻巨噬细胞的表型8。
一些研究表明，B细胞在心肌适应损伤的背景下起着重要作用8，9，10，11，12，13。B细胞在受伤的心脏中短暂积累，可能是通过CXCL13-CXCR5依赖机制11，13。从那里，B细胞通过几种机制促进不良心脏重塑，包括细胞因子介导的单核细胞募集9，12。此外，B细胞可以产生针对心脏蛋白的抗体，这些抗体可以通过几种机制促进心脏损伤的扩展和不良心脏重塑14，15，16，17，18，19，20，21，22，23，24，25.B细胞还可以通过分泌IL-10对受伤的心脏发挥保护作用10。
随着研究B细胞在幼稚和受伤心脏中的作用的小组数量的增加，定义共享协议以正确量化和评估心肌B细胞变得越来越重要，从而避免已经开始出现在文献中的不一致。事实上，据报道，B细胞都是啮齿动物心脏中最常见的免疫细胞之一7，并且其患病率明显低于骨髓细胞26，27，或者相当罕见28。同样，有几组人描述急性缺血性心肌损伤后心肌B细胞数量增加7、9、13，但有一组报告受损心肌B细胞数量没有变化29。对心脏免疫细胞的研究很少给出灌注条件的细节，对消化条件也没有达成共识。由于在啮齿动物心脏中，很大一部分B细胞在血管内，并且从心肌中提取免疫细胞高度依赖于所使用的消化方法，因此文献中报道的差异可能是器官灌注和组织消化差异的结果。
这里介绍的是一种基于流式细胞术的鼠心肌B细胞定量的详细方法，该方法通过优化灌注和消化条件来最大限度地提高B细胞回收率，并允许区分血管内与血管外心肌B细胞6。该协议是对区分血管内和间质免疫细胞的其他类似方案的改编和优化28，30，31。
在该协议中，我们将心肌灌注标准化，以消除漂浮在血管内空间中的B细胞，而无需去除粘附在微血管内皮上的生物学相关B细胞。此外，基于先前描述使用静脉注射抗体来区分血管内和间质免疫细胞的方案32，并利用B细胞表达表面标志物B22033的事实，我们演示了如何通过在动物处死和心脏灌注前立即血管内注射B220特异性抗体来区分血管内和血管外心肌B细胞。该协议与任何有兴趣包括幼稚和受伤心脏中心肌B细胞分析的科学家的研究相关。该协议的广泛实施将减少研究小组之间的不一致，将允许分析血管内和血管外心肌B细胞库的变化，从而促进心脏免疫学领域发现的进步。
总之，该协议代表了一种优化的工作流程，可通过流式细胞术量化和分析心肌B细胞，同时区分位于血管外空间和血管内间隙的细胞。
Protocol
本手稿中描述的所有实验均在约翰霍普金斯大学医学院IACUC的批准下进行。 1. 准备工作 准备FACS缓冲液，如 表1中所述。 确保有足够的CO2 对动物实施安乐死。 准备解剖空间（放置工作台垫并将胶带和解剖工具放在附近）。 标记15 mL试管，在每个试管中放入3 mL含钙和镁的HBSS（参见材料表）并将它们放…
Representative Results
采集完成并收集所有事件后，应根据标准流式细胞术实践分析数据。分析的重点将根据每个实验的个别目标而有所不同。在这种情况下，进行血管内和血管外B细胞的定量，并将其表示为每毫克组织的细胞数。 使用光谱细胞仪时，建议从初始设门开始分析，以去除大小不在免疫细胞对应范围内的碎片，然后去除活死染色+信号。这允许检测对应于白细胞的CD45阳性细胞的清洁群?…
Discussion
越来越多的证据表明，B细胞在心肌生理学和心肌重塑/适应损伤的背景下起着重要作用7，8，9，10，11，12，13，36。流式细胞术是研究任何组织中免疫细胞群的绝佳工具，几乎是任何专注于心脏中B?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究由NHLBI授予Luigi Adamo的5K08HLO145108-03和1R01HL160716-01资助。
用于开展这项研究的Aurora流式细胞仪由NIH资助S10OD026859资助。我们感谢JHU Ross流式细胞术核心的支持。
Materials
| Alexa Fluor 700 anti-mouse/human CD11b Antibody | 101222 | BioLegend | 100 µg 200 µL |
| (CellTreat 29481) Cell Strainer, 40 µm, Blue | QBIAP303 | Southern Labware | |
| 0.5 mL Natural Microcentrifuge Tube | 1605-0000 | SealRite, USA Scientific | |
| 0.9% Sodium Chloride Injection, USP | 114-055-101 | Quality Biological | 0.90% |
| 1.5 mL Natural Microcentrifuge Tube | 1615-5500 | SealRite, USA Scientific | |
| 10 µL Graduated TipOne Filter Tips | 11213810 | USA Scientific | |
| 1000 µL Graduated TipOne Filter Tips | 11267810 | USA Scientific | |
| 15 mL Centrifuge Tube, Plug Seal Cap, Polypropylene, RNase-/DNase-free | 430052 | Corning | |
| 1-Way Stop Valve, Polycarbonate | SVPT951 | ECT Manufacturing | |
| 2,2,2-Tribromoethanol | T48402 | Sigma-Aldrich | |
| 200 µL Graduated TipOne Filter Tips | 11208810 | USA Scientific | |
| 3-Way Stop Valve, Polycarbonate | SVPT953 | ECT Manufacturing | |
| 5 mL Polystyrene Round-Bottom Tube, 12 x 75 mm style | 352054 | Falcon, a Corning Brand | |
| 50 mL Centrifuge Tube, Plug Seal Cap, Polypropylene, RNase-/DNase-free | 430290 | Corning | |
| ACK (Ammonium-Chloride-Potassium) Lysing Buffer | 118-156-101 | Quality Biological | Osmolality: 290 + or -5% mOsm/Kg H20 |
| Adapter 4x50ml, for 250 mL rectangular bucket in Rotor A-4-63 | 5810759005 | Eppendorf | |
| Adapter for 15 mL Centrifuge Tubes, 9 Tubes per Adapter, Conical Bottom for use with Rotor Model A-4-62 | 22638289 | Eppendorf | |
| Adapter for 15 round-bottom tubes 2.6 – 7 mL, for 250 mL rectangular bucket in Rotor A-4-62 | 22638246 | Eppendorf | |
| Aluminum Foil 12 in x 75' Roll .0007 | UPC 109153 | Reynolds Wrap | |
| Anesthesia Induction Chamber – Mouse | RWD-AICMV-100 | Conduct Science | |
| BD Luer Slip Tip Syringe with attached needle 25 G x 5/8 in., sterile, single use, 1 mL | 309626 | BD Becton, Dickinson and Company | |
| Brandzig Ultra-Fine Insulin Syringes 29G 1cc 1/2" 100-Pack | CMD 2613 | Brandzig | |
| Brilliant Violet 421 anti-mouse CD19 Antibody | 115537 | BioLegend | 50 µg/mL |
| CAPS for Flow Tubes w/strainer mesh 35 µm, Dual position for 12 x 75 mm tubes, sterile | T9009 | Southern Labware | |
| Carbon Dioxide USP E CGA 940 | CD USPE | AirGas USA | |
| Cole-Parmer Essentials Low-Form Beaker, Glass, 500 mL | UX-34502-46 | Cole-Parmer | |
| Collagenase 2 | LS004176 | Sigma-Aldrich | |
| Connector brass chrome plated 1/4" female NPT x 1/4" barb | Y992611-AG | AirGas USA | |
| Cytek Aurora Flow Cytometer | Cytek Biosciences | ||
| Diss 1080 Nipple 1/4 BARB CP | M-08-12 | AirGas USA | |
| DNase I – 40,000 U | D4527 | Sigma-Aldrich | |
| Easypet 3 – Electronic Pipette Controller | 4430000018 | Eppendorf | |
| Electronic Balance, AX223/E | 30100606 | Ohaus Corp. | |
| Eppendorf 5810R centrifuge | 5810R | Eppendorf | |
| Eppendorf Research plus 1-channel variable pipettes | Eppendorf | ||
| FlowJo 10.8.1 | BD Becton, Dickinson and Company | ||
| GLACIERbrand, triple density Ice Pan (IPAN-3100) | Z740287 | Heathrow Scientific | |
| HBSS (1x) – Ca2+ [+] Mg2+ [+] | 14025076 | gibco | 1x |
| Hyaluronidase | H3506 | Sigma-Aldrich | |
| Kelly Hemostats, Straight | 13018-14 | Fine Science Tools | |
| Luer Slip Syringe sterile, single use, 20 mL | 302831 | BD Becton, Dickinson and Company | |
| M1 Adj. Reg 0-100 PSI/CGA940 | M1-940-PG | AirGas USA | |
| McKesson Underpads, Moderate | 4033-CS150 | McKesson | |
| Navigator Multi-Purpose Portable Balance | NV2201 | Ohaus Corp. | |
| PBS pH 7.4 (1X) Ca2+ [-] Mg2+ [-] | 10010023 | gibco | 1x |
| PE anti-mouse/human CD45R/B220 Antibody | 103208 | BioLegend | 200 µg/mL |
| PerCP/Cyanine5.5 anti-mouse CD45 Antibody | 103132 | BioLegend | 100 µg 500 uL |
| Petri dish, Stackable 35 mm x 10 mm Sterile Polystyrene | FB0875711YZ | Fisher Scientific | |
| Pkgd: Diss 1080 Nut/CO2/CO2-02 | M08-1 | AirGas USA | |
| Powerful 6 Watt LED Dual Goose-Neck Illuminator | LED-6W | AmScope | |
| PrecisionGlide Needle 25 G x 5/8 (0.5 mm x 16 mm) | 305122 | BD Becton, Dickinson and Company | |
| Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block) Clone 2.4G2 (RUO) | 553141 | BD Becton, Dickinson and Company Biosciences | 0.5 mg/mL |
| R 4.1.1 | The R Foundation | ||
| Razor Blades | 9501250000 | Accutec Blades Inc | |
| Regulator analytical two stage 0-25 psi delivery CGA320 3500 psi inlet | Y12244A320-AG | AirGas USA | |
| Rotor A-4-62, incl. 4 x 250 mL rectangular buckets | Rotor A-4-62 | Eppendorf | |
| Serological pipette, plugged, 10 mL, sterile, non-pyrogenic/endotoxin-free, non-cytotoxic, 1 piece(s)/blister | 86.1254.001 | Sarstedt AG & Co KG | |
| Sigma label tape | L8394 | Sigma-Aldrich | |
| SpectroFlo 3.0.0 | Cytek Biosciences | ||
| Spex VapLock Luer Fitting, PP, Straight, Male Luer Lock x 1/8" Hose Barb; 1/EA | MTLL230-6005 | Spex | |
| Std Wall Lab Tubing, Size S2, Excelon, 1/8" ID x 3/16" OD x 1/32" Wall x 50' Long | CG-730-003 | Excelon Laboratory | |
| Syringe PP/PE without needle, 3 mL | Z683566 | Millipore Sigma | |
| Syringe pump | 55-1199 (95-240) | Harvard Apparatus | |
| Thomas 3-Channel Alarm Timer TM10500 | 9371W13 | Thomas Scientific | |
| Tube Rack, 12 positions, 6 for 5.0 mL and 15 mL tubes and 6 for 25 mL and 50 mL tubes, polypropylene, numbered positions, autoclavable | 30119835 | Eppendorf | |
| Tube Rack, 12 positions, for 5.0 mL and 15 mL tubes, polypropylene, numbered positions, autoclavable | 30119827 | Eppendorf | |
| TYGON R-3603 Laboratory Tubing, I.D. × O.D. 1/4 in. × 3/8 in. | T8913 (Millipore Sigma) | Tygon, Saint-Gobain | |
| Vortex-Genie 2 | SI-0236 | Scientific Industries, Inc. | |
| VWR Dissecting Forceps with Guide Pin with Curved Tips | 89259-946 | Avantor, by VWR | |
| VWR Dissecting Scissors, Sharp Tip, 4½" | 82027-578 | Avantor, by VWR | |
| VWR Incubating Orbital Shaker, Model 3500I | 12620-946 | Avantor, by VWR | |
| Zombie Aqua Fixable Viability Kit | 423102 | BioLegend |
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Cite This Article
Bermea, K. C., Rousseau, S. T., Adamo, L. Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells. J. Vis. Exp. (186), e64344, doi:10.3791/64344 (2022).