Summary

基于流式细胞术的心肌B细胞定量和分析

Published: August 17, 2022
doi:

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34

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89,10111213。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1113。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912。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14,15,1617,1819,20,21,2223,2425.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、913,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283031

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7891011,121336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?…

Divulgazioni

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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Citazione di questo articolo
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).

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