体外犬中性粒细胞胞外陷阱的形成: 荧光显微镜动态定量分析
Summary
我们描述了从全血中分离犬中性粒细胞的方法, 用荧光显微镜观察活中性粒细胞的网状形成。还描述了使用免疫荧光显微镜定量分析网络形成和瓜组蛋白 H3 (citH3) 表达的协议。
Abstract
为了应对入侵的病原体, 中性粒细胞释放中性粒细胞胞外陷阱 (网), 它们是用组蛋白和抗菌蛋白修饰的 DNA 胞外网络。脓毒症期间过度的净形成 (NETosis) 和 citH3 释放与小鼠和人类多器官功能障碍和死亡有关, 但其对狗的影响是未知的。本文介绍了一种从全血中分离犬中性粒细胞的方法, 以观察和定量 NETosis。由葡聚糖沉淀产生的富含白细胞的等离子体, 通过商业上可用的密度梯度分离介质和细胞计数和生存能力测试收集的颗粒分离。为了观察实时 NETosis 在活中性粒细胞, 细胞 permeant 和细胞 impermeant 荧光核酸染色添加到中性粒细胞激活, 无论是由脂多糖 (LPS) 或佛波 12-酯 13-醋酸酯 (PMA)。随着时间的推移, 荧光显微镜观察到核形态和网状形成的变化。体外NETosis 进一步的特点是定位的无细胞 DNA (cfDNA), 髓 (过氧化物酶) 和瓜组蛋白 H3 (citH3) 使用改良的双 immunolabelling 协议。为了客观地量化网络形成和 citH3 表达, 使用荧光显微镜, 网和 citH3-positive 细胞是量化的盲目的方式使用可用的软件。该技术是评价犬中性粒细胞体外能力 NETosis 的一种特殊检测方法。
Introduction
中性粒细胞是短寿命的颗粒, 负责初步防御入侵病原体。嗜中性粒细胞被招募到感染部位, 通过吞噬、脱粒和产生活性氧 (ROS)1消除微生物。在细菌或内毒素存在的情况下, 中性粒细胞释放中性粒细胞胞外陷阱 (网), 由细胞外染色质修饰的组蛋白和颗粒蛋白, 如弹性蛋白酶和髓 (过氧化物酶)2。虽然蚊帐具有不可缺少的抗菌性能, 但越来越多的实验和临床证据表明, 脓毒症的过度过度网络形成可能导致多器官功能障碍和死亡3,4,5,6。
由于蚊帐可能在犬类中起到类似的病理生理作用, 预防或减少网状形成的治疗性干预可以作为脓毒症动物的新治疗策略。因此, 需要一种可靠的技术来评估和量化狗的 NETosis 和网络成分。包括无细胞 DNA (cfDNA) 和核在内的净成分在犬中性粒细胞和7、8、9的临床犬血浆中得到了评价。通过荧光化验, Goggs 和 Letendre 发现, 脓毒症犬的 cfDNA 水平比健康犬高8。虽然这些技术是高度客观和定量的, 测量 cfDNA 和核作为标记的 NETosis 是非特异的, 因为它们可以从坏死细胞以外的 NETosing 中性粒细胞。在这里, 我们描述了一种技术, 利用荧光显微镜检查活 NETosing 中性粒细胞的行为。我们还详细介绍了一种改进的协议, 使用双 immunolabeling 主观量化的网及其组成部分, 如髓内过氧化物酶和 citH3 在犬中性粒细胞10。
Protocol
Representative Results
Discussion
我们在这里提出一个协议, 以观察细胞 permeant 染料和细胞 impermeant 染料的活犬中性粒细胞细胞核构象和 cfDNA 释放的变化。该方法的主要优点是, 它允许在无细胞固定的情况下, 通过高分辨率显微术实时检测网络形成, 为观察体外网状形成提供了一个简单而有价值的工具。由于这种化验不需要抗体检测的净成分, 它是理想的研究 NETosis 在物种特定的抗体有限的可用性。利用这一方法, 作者证明, …
Declarações
The authors have nothing to disclose.
Acknowledgements
相应的作者由莫里斯动物基金会 (D15CA-907) 资助。这项研究得到了加州大学戴维斯分校马健康中心和同伴动物健康中心 (2016-24 F) 的资助。作者想感谢 Geena 的帮助与数字和尼格海谭德阮为她的视频协助。
Materials
| Dextran from Leuconostoc spp. | Sigma | 31392 | Molecular weight 450,000 – 650,000 |
| Ficoll-Paque PLUS | GE Life Sciences | 17144002 | |
| Dulbecco’s Phosphate-Buffered Saline | ThermoFisher Scientific | A1285801 | With divalent cations |
| Dulbecco’s Phosphate-Buffered Saline | ThermoFisher Scientific | 14190136 | Without divalent cations |
| E. coli O55:B5 | InvivoGen | ||
| SYTOX Orange Fluorescent Nucleic Acid Stain | ThermoFisher Scientific | S11368 | 5 mM in DMSO; stains in cells with permeable membranes |
| SYTO Green 16 Fluorescent Nucleic Acid Stain | ThermoFisher Scientific | S7578 | 1 mM in DMSO; stains in cells with intact membranes |
| Surface-Amps NP-40 | Pierce | 28324 | |
| Poly-D-Lysine coated coverslips | neuVitro | H-12-pdl | 12 mm diameter |
| Anti-citrullinated histone H3 antibody | Abcam | Ab5103 | |
| Unconjugated goat anti-rabbit Fab fragments | Jackson ImmunoResearch | 111-007-003 | Specificity: IgG (H+L) |
| Anti- Myeloperoxidase antibody | Dako | A0398 | |
| 4,6-Diamidino-2-phenylin (DAPI) | Life Technologies Corporation | D1306 |
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