一套快速了解中性粒细胞功能的筛查技术
Summary
该协议具有一组中性粒细胞功能测定，可用作筛选方法，以涵盖来自不同信号通路的功能。该协议包括对细胞活力、纯度、活性氧产生、实时迁移、吞噬作用的初步和简单评估，以及中性粒细胞细胞外陷阱的初步建议。
Abstract
中性粒细胞被称为先天免疫反应的第一道防线之一，可以执行许多特定的细胞功能，例如趋化性、反向迁移、吞噬作用、细胞毒性酶和代谢物的脱颗粒，以及 DNA 作为中性粒细胞胞外陷阱 （NET） 的释放。中性粒细胞不仅自身具有严格调节的信号传导，而且还参与免疫系统其他成分的调节。由于新鲜的中性粒细胞是终末分化的、短暂的，并且个体之间差异很大，因此充分利用收集的样本非常重要。研究人员通常需要进行筛选分析，以评估可能受评估的特定条件影响的许多中性粒细胞功能的概述。为了满足这一需求，开发了一套遵循正常密度中性粒细胞单一分离过程的测试，寻求速度、全面性、成本和准确性之间的平衡。研究结果可用于推理和指导深入的后续研究。该过程可以在平均 4 小时内进行，包括评估细胞活力、活性氧 （ROS） 产生、实时迁移和载玻片上酵母的吞噬作用，留下足够的细胞用于更详细的方法，如组学研究。此外，该程序包括一种在通过光学显微镜观察快速全景染色后轻松观察NETs初步建议的方法，尽管缺乏特异性标记，但足以表明是否值得以这种方式进行进一步的努力。测试功能的多样性结合了测试之间的共同点，减少了分析时间和费用。该程序被命名为NeutroFun Screen，虽然有局限性，但它平衡了上述因素。此外，这项工作的目的不是一个明确的测试集，而是一个可以很容易地根据每个实验室的资源和需求进行调整的指南。
Introduction
中性粒细胞是人体血液中最丰富的先天免疫细胞，已知在感染和炎症中起主要作用，是到达组织损伤部位的第一反应者1.近年来，人们越来越认识到中性粒细胞在各种疾病和支持体内平衡方面发挥的关键作用2。中性粒细胞不仅自身具有严格调节的信号传导，而且还参与免疫系统其他成分的调节3,4,5。因此，在许多研究环境中，研究中性粒细胞及其许多不寻常的细胞功能，如趋化性、反向迁移6、吞噬作用7、呼吸爆发8 和中性粒细胞胞外陷阱 （NETs） 的释放7，在许多研究环境中势在必行，需要评估由所分析的特定条件引发的潜在中性粒细胞功能、形态或分子变化。
新鲜分离的中性粒细胞是终末分化的、短命的、高动态的和容易激活的9.然而，尚未实现不影响中性粒细胞反应的有效储存方法，因此很难进行必须不间断的多种检测。此外，当需要对中性粒细胞进行广泛和初步评估时，先前描述的功能分析10,11基于需要细胞术和/或荧光染色的测定可能不是可行的选择。
为了解决这些问题，该协议描述了一组可以在单个分离过程之后进行的测试，包括评估细胞活力，活性氧（ROS）产生，实时迁移和 酿酒酵母的吞噬作用，其结果可用于推理深入的后续研究。该程序名为NeutroFun Screen，旨在包含除脱颗粒以外的主要效应器活动，并且可以在平均4小时内完成，包括1小时的激活。此外，剩余的细胞可用于更详细的方法，如组学研究。这种方法的优点在于它在速度、全面性、成本和准确性之间取得平衡。
此外，有一种方法可以很容易地观察到NET的初步建议，没有具体的标记，但足以表明是否值得朝这个方向进一步努力。测试功能的多样性旨在结合测试之间的共同点，减少分析时间和费用。该方法的主要目标是提供关于速度、全面性、成本和准确性的平衡、功能分析，从而可以概述中性粒细胞的反应，使其成为研究新刺激对正常密度中性粒细胞影响的有用的第一步。
Protocol
所有实验都严格遵循巴西利亚大学机构审查委员会制定的伦理准则（流程13364819.0.0000.5558），并且样本通过代码进行识别，以确保捐赠者的匿名性。这些细胞是从年龄在 18-35 岁之间的正常健康男性供体中获得的，他们签署了知情同意书并符合以下资格标准：非吸烟者/电子烟使用者，没有慢性健康状况，并且在过去 14 天内没有炎症病史。 1. 采血 无菌地将 0…
Representative Results
本研究中使用的基于密度的分离方法（图1）符合所提出的实验标准。从该方法获得的中性粒细胞参数包括活力≥98%、纯度≥94%和细胞产量≥1.5 x 107，筛选试验未检测到活化。分离 PMN 的两个相关步骤是抗凝和去除红细胞。在密度梯度上分层之前，将抗凝血管或注射器保持在轻轻摇晃状态，并选择红细胞去除方法来防止活化和污染，可能会影响实验产量和可重复性。</…
Discussion
中性粒细胞是高度动态和反应性的细胞，寿命短，尚不能冷冻保存19，这使得对其生物学的研究具有挑战性。因此，必须遵循谨慎的步骤来获得可存活、富集和静息的中性粒细胞11,20。本研究采用了基于密度的隔离技术，强调温和和最小的操作，以及在活化步骤之前使用低温。此外，血液处理必须在静脉穿刺后 30 分钟内进行，并在室温…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢以下资助机构：FAPDF、CNPq、CAPES、UnB、FINEP 和 FINATEC。
Materials
| CIM-Plate 16 | Agilent | 5665825001 | |
| CLARIOstar Plate Reader | BMG LABTECH | US Patent Number 9,733,124 Product details: MARS Data Analysis Software |
|
| Dimethyl sulfoxide | Dinâmica | 1582 | |
| DNAse I | Sigma – Aldrich | DN 25 | |
| Ethylenediaminetetraacetic acid disodium salt dihydrate | Sigma – Aldrich | E5134 | |
| Fast panoptic stain | Laborclin | 620529 | |
| Glass slide | Exacta | 7102 | |
| Hank’s Balanced Salt Solution with calcium, with magnesium, without phenol red. | Sigma – Aldrich | 55037C | |
| Hank’s Balanced Salt Solution without calcium chloride, magnesium sulfate and sodium bicarbonate. | Sigma – Aldrich | H4641 | |
| Heparin | Blau | 7896014655229 | |
| Laminar flow cabinet | Veco | VLFS-12 | |
| Microscope | Zeiss | 415501-0101-002 | Product details: Primostar 1 |
| Mixing Block | BIOER | MB-102 | |
| Neubauer improved bright-lined | New Optik | 1110000 | |
| N-formyl-methionyl-leucyl-phenylalanine | Sigma – Aldrich | F3506 | |
| Nitroblue tetrazolium | Neon | CAS 298-83-9 | |
| Percoll | Cytiva | 17089101 | separation media |
| Phorbol 12-myristate 13-acetate | Sigma – Aldrich | P8139 | |
| Phosphate buffered saline tablet | Sigma – Aldrich | P4417 | |
| ROTOFIX 32 A | Hettich | 1206 | |
| Saccharomyces cerevisiae | Fleischmann | ||
| Safranin | Sigma – Aldrich | 50240 | |
| Sodium dodecyl sulfate | Cytiva | 17-1313-01 | |
| Sonicator | Qsonica | Q125 | |
| Trypan blue solution | Vetec | C.I. 23850 | |
| Vortex Genie 2 | Scientific Industries, Inc. | 0K-0500-902 | |
| xCELLigence Real-Time Cell Analysis (RTCA) DP (dual purpose) | Agilent | 380601050 | Product details: RTCA system composed of detection hardware, cell plates and software |
References
- Nauseef, W. M., Borregaard, N. Neutrophils at work. Nature Immunology. 15 (7), 602-611 (2014).
- Groeneweg, L., Hidalgo, A. Emerging roles of infiltrating granulocytes and monocytes in homeostasis. Cellular and Molecular Life Sciences. 77 (19), 3823-3830 (2020).
- Rosales, C., Lowell, C. A., Schnoor, M., Uribe-Querol, E. Neutrophils: their role in innate and adaptive immunity 2017. Journal of Immunology Research. 2017, 9748345 (2017).
- Castro, M., et al. Proteome analysis of resting human neutrophils. Protein & Peptide Letters. 13 (5), 481-487 (2006).
- Li, Y., et al. The regulatory roles of neutrophils in adaptive immunity. Cell Communication and Signaling. 17, 147 (2019).
- de Oliveira, S., Rosowski, E. E., Huttenlocher, A. Neutrophil migration in infection and wound repair: going forward in reverse. Nature Reviews Immunology. 16 (6), 378-391 (2016).
- Burn, G. L., Foti, A., Marsman, G., Patel, D. F., Zychlinsky, A. The neutrophil. Immunity. 54 (7), 1377-1391 (2021).
- El-Benna, J., et al. Priming of the neutrophil respiratory burst: role in host defense and inflammation. Immunological Reviews. 273 (1), 180-193 (2016).
- Castro, M. S., Cilli, E. M., Fontes, W. Combinatorial synthesis and directed evolution applied to the production of alpha-helix forming antimicrobial peptides analogues. Current Protein & Peptide Science. 7 (6), 473-478 (2006).
- Mihaila, A. C., et al. Transcriptional profiling and functional analysis of N1/N2 neutrophils reveal an immunomodulatory effect of S100A9-blockade on the pro-inflammatory N1 subpopulation. Frontiers in Immunology. 12, 708770 (2021).
- Kuhns, D. B., Priel, D. A. L., Chu, J., Zarember, K. A. Isolation and functional analysis of human neutrophils. Current Protocols in Immunology. 111 (1), 7-23 (2015).
- Paulíková, E., Kociková, A., Sabol, M. Modification of a panoptic method of staining isolated cells. Bratislavske Lekarske Listy. 94 (12), 638-640 (1993).
- Strober, W. Trypan blue exclusion test of cell viability. Current Protocols in Immunology. 111 (1), 1-3 (2015).
- Libério, M. S., et al. Anti-proliferative and cytotoxic activity of pentadactylin isolated from Leptodactylus labyrinthicus on melanoma cells. Amino Acids. 40 (1), 51-59 (2011).
- Cano, P. M., Vargas, A., Lavoie, J. P. A real-time assay for neutrophil chemotaxis. BioTechniques. 60 (5), 245-251 (2016).
- Stefanowicz-Hajduk, J., Adamska, A., Bartoszewski, R., Ochocka, J. R. Reuse of E-plate cell sensor arrays in the xCELLigence Real-Time Cell Analyzer. BioTechniques. 61 (3), 117-122 (2016).
- Björkstén, B., Nyström, K., Lindqvist, B. The nitroblue tetrazolium (NBT) test in endemic benign (epidemic) nephropathy. Acta Medica Scandinavica. 199 (1-6), 147-150 (1976).
- Aquino, E., et al. Proteomic analysis of neutrophil priming by PAF. Protein & Peptide Letters. 23 (2), 142-151 (2016).
- Blanter, M., Gouwy, M., Struyf, S. Studying neutrophil function in vitro: cell models and environmental factors. Journal of Inflammation Research. 14, 141-162 (2021).
- Hsu, A. Y., Peng, Z., Luo, H., Loison, F. Isolation of human neutrophils from whole blood and buffy coats. Journal of Visualized Experiments. (175), e62837 (2021).
- Moghadam, Z. M., Henneke, P., Kolter, J. From flies to men: ROS and the NADPH oxidase in phagocytes. Frontiers in Cell and Developmental Biology. 9, 628991 (2021).
- Pattan, S. S., Bhat, K. G., Pattar, G. D., Kuntagi, M. Comparison of three different techniques for isolation of neutrophils from blood and their utility in performing nitroblue tetrazolium test. International Journal of Basic and Applied Physiology. 8 (1), 41 (2019).
- Gooty, J. R., Shashirekha, A., Guntakala, V. R., Palaparthi, R. Estimation of phagocytic activity of polymorphonuclear leukocytes in chronic and aggressive periodontitis patients with nitroblue tetrazolium test. Journal of Indian Society of Periodontology. 23 (4), 316 (2019).
- Langer, S., et al. Clinical and laboratory profiles of 17 cases of chronic granulomatous disease in north India. Indian Journal of Hematology and Blood Transfusion. 37 (1), 45-51 (2021).
- Oualha, R., et al. Infection of human neutrophils with Leishmania infantum or Leishmania major strains triggers activation and differential cytokines release. Frontiers in Cellular and Infection Microbiology. 9, 153 (2019).
- Zilinskas, J., Zekonis, J., Zekonis, G., Valantiejiene, A., Periokaite, R. The reduction of nitroblue tetrazolium by total blood in periodontitis patients and the aged. Stomatologijal. 9 (4), 105-108 (2007).
- Benov, L. Improved formazan dissolution for bacterial MTT assay. Microbiology Spectrum. 9 (3), e01637 (2021).
- Chen, Y., Junger, W. G. Measurement of oxidative burst in neutrophils. Methods in Molecular Biology. 844, 115-124 (2012).
- Richardson, M. P., Ayliffe, M. J., Helbert, M., Davies, E. G. A simple flow cytometry assay using dihydrorhodamine for the measurement of the neutrophil respiratory burst in whole blood: comparison with the quantitative nitrobluetetrazolium test. Journal of Immunological Methods. 219 (1-2), 187-193 (1998).
- Jancinová, V., et al. The combined luminol/isoluminol chemiluminescence method for differentiating between extracellular and intracellular oxidant production by neutrophils. Redox Report. 11 (3), 110-116 (2006).
- Nosál, R., et al. Pharmacological intervention with oxidative burst in human neutrophils. Interdisciplinary Toxicology. 10 (2), 56-60 (2017).
- Mol, S., et al. Efficient neutrophil activation requires two simultaneous activating stimuli. International Journal of Molecular Sciences. 22 (18), 10106 (2021).
- Schneider, L., et al. Flow cytometry evaluation of CD14/CD16 monocyte subpopulations in systemic sclerosis patients: a cross sectional controlled study. Advances in Rheumatology. 61 (1), 27 (2021).
- Akin, E., Pelen, N. N., Tiryaki, I. U., Yalcin, F. Parameter identification for gompertz and logistic dynamic equations. PLoS One. 15 (4), e0230582 (2020).
- Guy, J. B., et al. Evaluation of the cell invasion and migration process: A comparison of the video microscope-based scratch wound assay and the boyden chamber assay. Journal of Visualized Experiments. (129), e56337 (2017).
- Brinkmann, V., et al. Neutrophil extracellular traps kill bacteria. Science. 303 (5663), 1532-1535 (2004).
- de Bont, C. M., Koopman, W. J. H., Boelens, W. C., Pruijn, G. J. M. Stimulus-dependent chromatin dynamics, citrullination, calcium signalling and ROS production during NET formation. Biochimica et Biophysica Acta. Molecular Cell Research. 1865, 1621-1629 (2018).
- Masuda, S., et al. Measurement of NET formation in vitro and in vivo by flow cytometry. Cytometry Part A. 91 (8), 822-829 (2017).
- Zharkova, O., et al. A flow cytometry-based assay for high-throughput detection and quantification of neutrophil extracellular traps in mixed cell populations. Cytometry Part A. 95 (3), 268-278 (2019).
- Hosseinnejad, A., et al. DNase I functional microgels for neutrophil extracellular trap disruption. Biomaterials Science. 10 (1), 85-99 (2022).
- Chrysanthopoulou, A., et al. Neutrophil extracellular traps promote differentiation and function of fibroblasts. The Journal of Pathology. 233 (3), 294-307 (2014).
- Tong, M., Abrahams, V. M. Visualization and quantification of neutrophil extracellular traps. Methods in Molecular Biology. 2255, 87-95 (2021).
- Santana, C. J. C., et al. Biological properties of a novel multifunctional host defense peptide from the skin secretion of the chaco tree frog, boana raniceps. Biomolecules. 10 (5), 790 (2020).
- Murphy, M. P., et al. Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. Nature Metabolism. 4 (6), 651-662 (2022).
- Boero, E., et al. Use of flow cytometry to evaluate phagocytosis of staphylococcus aureus by human neutrophils. Frontiers in Immunology. 12, 635825 (2021).
- Karsten, C. B., et al. A versatile high-throughput assay to characterize antibody-mediated neutrophil phagocytosis. Journal of Immunological Methods. 471, 46-56 (2019).
- Smirnov, A., Solga, M. D., Lannigan, J., Criss, A. K. Using imaging flow cytometry to quantify neutrophil phagocytosis. Methods in Molecular Biology. 2087, 127-140 (2020).
Cite This Article
Souza Luz, I., Takaya, R., Gonzaga Ribeiro, D., Sales Silva, N., Fontes, L., Castro, M. S., Fontes, W. A Set of Screening Techniques for a Quick Overview of the Neutrophil Function. J. Vis. Exp. (204), e65329, doi:10.3791/65329 (2024).