小鼠特拉切斯刷细胞的分离与定量评价
Summary
刷细胞是罕见的胆碱能化学感觉上皮细胞发现在天真的小鼠气管。由于其数量有限,其功能在气道免疫和改造中的作用的体外评估具有挑战性。我们描述了一种通过流式细胞测定分离气管刷细胞的方法。
Abstract
流毛刷细胞是胆碱能化学感觉上皮细胞,准备将信号从气道流明传输到免疫和神经系统。它们是化学感觉上皮细胞家族的一部分,包括肠道粘液中的突触细胞、气管中的刷细胞以及鼻黏液中的单独化学感觉和微绒细胞。不同上皮腔的切觉细胞共享关键的细胞内标记和核心转录特征,但也表现出显著的转录异质性,可能反映局部组织环境。需要从单细胞悬浮液中分离气管刷细胞,以详细定义这些稀有上皮细胞的功能,但它们的分离具有挑战性,这可能是由于气管刷细胞和神经末梢之间的密切相互作用或由于气道特异性的紧密和粘附结的组成。在这里,我们描述了从小鼠气管上皮分离刷细胞的过程。该方法基于气管上皮从亚粘液中的初始分离,允许随后用木瓜进行上皮板的较短孵育。此过程为流动细胞分和可行气管刷细胞的功能分析提供了快速、方便的解决方案。
Introduction
刷细胞属于一类化学感官上皮细胞,其特征是苦味受体的表达和味蕾细胞中发现的味觉受体转导机制。与味蕾细胞不同,化学感官上皮细胞分散在上皮表面,被称为鼻上皮1、2、气管中的微微神经质细胞。3,4,和肠中的突子细胞5,6。表达苦味受体和苦味转导机制的皮下细胞也存在于尿道7、8和听觉管9中。气道刷细胞在神经生成和免疫气道反应中具有独特的功能。它们是乙酰胆碱产生的化学感觉细胞,在用苦化合物和细菌代谢物(如法定感物质10)激活时引起保护性呼吸反射。气道刷细胞也是IL-25的主要气道上皮来源,它调节气道3中的气过敏原-2型炎症。
低气道刷细胞的完整转录组及其对环境刺激的反应的表征,由于在气管上皮中数量少,且大支气管10以外的数量非常有限。用于从肠道上皮分离化学感觉细胞的技术并没有从气管中产生比例高的数字,可能是因为神经末梢为10或其他的气管刷细胞的亲密接触呼吸粘部中的组织特异性因子,如粘附物和紧密结蛋白的组成。最近关于单细胞RNA测序分析成功分离高数量的气管刷细胞的报告,要么采用2小时培养,要么用帕金孵育,要么用18小时孵育11,12。由于使用消化酶的较长孵育期会降低细胞活力,并改变消化组织13细胞的转录轮廓,这可能偏向其他化学感觉上皮种群的比较分析。
在这里,我们报告一种用于RNA测序的气管刷细胞分离方法。用高剂量去巴西气管治疗将上皮从亚粘管中分离出来。随后消化上皮片与木瓜,允许这种结构细胞的出色地恢复。
Protocol
在进行以下实验之前,确保所有动物护理使用和规程都得到机构动物护理和使用委员会 (IACUC) 的批准,并符合国家研究理事会的”护理和使用指南”。实验室动物”( 第8版,2011年)和《大起百万人指南》。下文所述的所有程序都经过布里格姆和妇女医院的机构动物护理和使用委员会的审查和批准。 1. 试剂制备 制备脱脂消化溶液,这是一种PBS溶液,含有16U/mL消?…
Representative Results
这个程序已经成功地实现分离气管刷细胞的RNA测序3。用2步方案分离气管和消化组织后,在排除带有PI的死细胞后,用荧光标记CD45和EpCAM收集并染色细胞。在基于正向和侧散射特性门控双精度点后,我们将画笔单元定义为 CD45 的低/负、EpCAM 的正值和 eGFP 的正数(图 2A)。刷细胞按流动细胞测定表示CD45低/负细胞的±0.16-0.42%…
Discussion
我们发现,高剂量消血治疗40分钟,然后短的木瓜治疗(30分钟)的组合为气管消化和刷细胞分离提供了最佳方案。与替代协议相比,这种组合既避免了广泛的消化,又能产生最高产量的刷细胞。
虽然肺消化提取造血细胞传统上依赖于温和的消化酶,如胶原酶IV15,但分离上皮细胞需要更复杂的方案。上皮细胞的单细胞悬浮体更难实现,因为紧密和粘附结将它们结合在彼此…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢布里格姆和妇女人类免疫学中心流动核心的亚当·奇币,感谢他在流式细胞分型方面的帮助。这项工作得到了国家卫生研究院资助R01 HL120952(N.A.B.),R01 AI134989(N.A.B),U19 AI095219(N.A.B., L.G.B) 和 K08 AI132723 (L.G.B),由美国过敏、哮喘和免疫学学会 (AAAAI)/ 美国肺过敏呼吸疾病奖(N.A.B.),由AAAAI基金会教师发展奖(L.G.B.),史蒂文和朱迪·凯青年创新者奖(N.A.B.),乔伊斯琳·奥斯汀女医生职业发展基金(L.G.B.)颁发,维尼克家族的慷慨捐赠(L.G.B.)。
Materials
| Antibodies | |||
| Anti-GFP (Polyclonal goat Ig) | Abcam | cat# ab5450 | |
| APC anti-mouse CD326 (EpCAM) (G8.8) | Biolegend | cat#118214 | |
| APC Rat IgG2a, k isotype control | Biolegend | cat#400511 | |
| DAPI | Biolegend | cat#422801 | |
| Donkey anti-goat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Life Technologies/Molecular Probes | cat#A-11055 | |
| Normal Goat IgG | R&D Systems | cat#AB-108-C | |
| Pacific Blue anti-mouse CD45 (30F-11) | Biolegend | cat#103126 | |
| Pacific Blue Rat IgG2b, k isotype control | Biolegend | cat#400627 | |
| TruStain FcX (anti-mouse CD16/32) Antibody | Biolegend | cat#101320 | |
| Chemicals, Peptides, and Recombinant Proteins | |||
| Dispase | Gibco | cat# 17105041 | |
| DNase I | Sigma | cat# 10104159001 | |
| HEPES-Tyrode’s Buffer Without Calcium (10 mM HEPES, 135 mM NaCl, 2.8 mM KCl, 1 mM MgCl2, 12 mM NaHCO3, 0.4 mM NaH2PO4, 0.25% BSA, 5.5 mM Glucose. Prepared in 18.2 megohms water and filtered through 0.22 µm filter | Boston BioProducts | cat# PY-912 | |
| Tyrode’s Solution (HEPES-Buffered) 140 mM NaCl, 5 mM KCl, 25 mM HEPES, 2 mM CaCl2, 2 mM MgCl2 and 10 mM glucose. Prepared in 18.2 megohms water and filtered through 0.22 µm filter. ) | Boston BioProducts | cat# BSS-355 | |
| L-Cysteine | Sigma | cat# C7352 | |
| Leupeptin trifluoroacetate salt | Sigma | cat# L2023 | |
| Papain from papaya latex | Sigma | cat# P3125 | |
| Propidium iodide | Sigma | cat# P4170 | |
| Experimental Models: Organisms/Strains | |||
| ChATBAC-eGFP (B6.Cg-Tg(RP23-268L19-EGFP)2Mik/J) | The Jackson Laboratory | 7902 | |
| Equipment | |||
| LSM 800 with Airyscan confocal system on a Zeiss Axio Observer Z1 Inverted Microscope | Zeiss | ||
| LSRFortessa | BD | 647465 | |
| Disposable equipment | |||
| 1.5 mL sterile tubes | Thomas Scientific | 1157C86 | |
| 5 mL Poysterene Round-bottom Tube, 12 x 75 mm style | Falcon | 14-959-1A | |
| 50 mL Polypropylene conical tube, 30 x 115 mm style | Falcon | 352098 | |
| Feather Disposable Scalpel no.12 | Fisher Scientific | NC9999403 | |
| Petri dish, 100 x 15 mm Style | Falcon | 351029 | |
| Sterile cell strainer, 100 μm | Fisherbrand | cat#22363549 |
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Cite This Article
Ualiyeva, S., Yoshimoto, E., Barrett, N. A., Bankova, L. G. Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas. J. Vis. Exp. (148), e59496, doi:10.3791/59496 (2019).