利用流细胞测定人类白细胞测定阿斯珀吉鲁斯紫锥体烟曲的噬菌体
Summary
该协议提供了一种快速可靠的方法，用流动细胞测定人类原发性噬菌体定量测量阿苯甲基的邻苯二甲苯，并区分锥体从单纯的粘附到白细胞的邻形图。
Abstract
霉菌阿斯珀吉鲁斯熏蒸菌的侵入性肺感染对免疫功能低下的患者构成巨大威胁。吸入真菌锥体（孢子）通过先天单核细胞和/或中性粒细胞的噬菌体从人类肺肺泡。该协议提供了一种快速、可靠的测量方细胞化，使用氟素等氰酸酯 （FITC） 标记的锥体与人类白细胞共同孵育，随后与抗 FITC 抗体进行反染色，以便对内化和细胞粘附锥体进行鉴别。该协议的主要优点是其快速性，将测定与其他感兴趣的细胞标记的细胞学分析相结合的可能性，同时分析单个样本中的单核细胞和嗜中性粒细胞，以及其对其他细胞壁携带真菌或细菌的适用性。测定方位白细胞的百分比，为微生物学家评估病原体的毒性或比较病原体的野生型和突变体提供了手段，也为免疫学家提供了研究人类白细胞对抗病原体的能力的手段。
Introduction
侵入性肺动脉粥样硬化对免疫功能低下患者是一个很大的威胁，因为治疗方案有限，只有在早期诊断成功，导致高死亡率1。传染性病原体是阿塞珀吉鲁斯熏蒸剂的锥度（孢子），在大多数栖息地无处不在2。Conidia 被吸入，通过气道，最后可以进入肺肺泡。在免疫能力人类中，这些锥体由先天免疫细胞清除，如单核细胞或巨噬细胞和嗜中性粒细胞，它们吸收（噬菌体）并消化病原体3。当对宿主-病原体相互作用感兴趣时，噬菌体病对微生物学家和免疫学家也很重要。对抗测定，如白细胞和锥体的共同孵育，通常包括用荧光素或其衍生的荧光素等子素（FITC）标记孢子。使用显微镜，它很容易识别内化荧光锥体，并确定附加/粘附锥体，虽然这种方法是繁琐和现实地限于几百个细胞4。然而，在流式细胞学中，很容易在几分钟内分析数十万个细胞，对方位细胞和粘附性锥体的不同染色至关重要。因此，许多协议依靠Trypan Blue来淬火FITC荧光从粘附锥体5，6，7，8。另一种方法是利用溴化和FITC的荧光共振能量转移，从粘附的锥度9，10，11发射红色，而不是绿色荧光。如果有特定的抗体可用，就像某些细菌一样，细胞结合的颗粒可以直接染色12，13。
在这里，我们提出一个协议，通过采用异位素青霉素（APC）耦合抗FITC抗体，快速定量地评估带有PCC标签的人类白细胞的芬加菌锥体，以及孢子附着在细胞上和缺乏相互作用。该方法还允许同时对来自同一样品的单核细胞和嗜中性粒细胞的食细胞化进行进一步细胞标记的流动细胞学分析。
该协议可应用于真菌菌株的表征（例如，这里介绍的Mucorales属的几种Aspergillus和其他霉菌）及其突变体14和对噬菌体（如免疫功能低下个体的白细胞）的免疫学研究。
Protocol
Representative Results
Discussion
该协议提出了一种快速流动细胞学方法，用于测量A.熏蒸锥体与大量初级人类白细胞的相互作用，这在常见的显微协议中是不可能的。使用显微镜和手动计数内化锥体成像细胞非常麻烦，实际上只能对几百个细胞进行成像。流式细胞测量通过在几分钟内测量数千个细胞来克服这个问题。这两种方法共有的障碍是细胞内或细胞上的邻形同质体的区别。在显微镜中，染料钙氟白色常用于染色粘?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢皮娅·斯蒂尔夫人的出色技术援助。M. von Lilienfeld-Toal 由败血症控制和护理中心（德国联邦教育和卫生部、BMBF、FKZ 01E01002）和感染性生物研究园区（BMBF，FKZ 13GW0096D）提供支持。蒂恩戈克·迈·霍恩由耶拿微生物传播学院（德国福森斯格明舍夫特，FKZ 214/2）支持
Materials
| Adhesive foil | Brand | 701367 | |
| anti-CD14 V500 | BD Biosciences | 561391 | clone M5E2 |
| anti-CD45 BUV395 | BD Biosciences | 563792 | clone HI30 |
| anti-CD66b PerCP-Cy5.5 | BD Biosciences | 562254 | clone G10F5 |
| anti-FITC APC | ThermoFisher Scientific | 17-7691-82 | clone NAWESLEE |
| Cell culture plate, 12-well | Greiner Bio-one | 665180 | |
| Cell scraper | Bioswisstech | 800020 | |
| Cell strainer, 30 µm | Miltenyi Biotech | 130-098-458 | SmartStrainer |
| Cytometer | BD Biosciences | LSR Fortessa II, lasers: 488 nm (blue), 405 nm (violet), 355 nm (UV) and 640 nm (red) | |
| Detergent | Sigma Aldrich | P1379 | Tween 20, 0.01% in PBS |
| Drigalski spatula | Carl Roth | PC59.1 | |
| Ethylenediaminetetraacetic acid (EDTA) | Sigma Aldrich | ED3SS-500g | 2 mM in PBS |
| Erythrocyte lysis buffer | 0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA | ||
| Fetal Calf Serum (FCS) | Biochrom AG | S 0115 | 10% in RPMI 1640 |
| Fluorescein isothiocyanate (FITC) | Sigma Aldrich | F3651-100MG | 0.1 mM in Na2CO3 /PBS solution |
| Formaldehyd | Carl Roth | PO87.3 | Histofix |
| Malt agar (1.5%) | malt extract (40 g), yeast extract (4 g), agar (15 g), Aqua dest. (1 L), adjust pH to 5.7-6.0, sterilise at 121 °C for 35 minutes | ||
| Na2CO3 | Carl Roth | 8563.1 | 0.1 M in PBS |
| Petri dish | Greiner Bio-one | 633180 | |
| Phosphat Buffered Saline (PBS) | ThermoFisher Scientific | 189012-014 | without Calcium, without Magnesium |
| RPMI 1640 | ThermoFisher Scientific | 61870010 | RPMI 1640 Medium, GlutaMAX Supplement |
| Rotator | Miltenyi Biotech | 130-090-753 | MACSmix Tube Rotator |
| Round-bottom tube, 7.5 mL | Corning | REF 352008 | |
| Software for data acquisition and analysis | BD Biosciences | FACSDiva 8.0 | |
| V-bottom plate, 96 well | Brand | 781601 | untreated surface |
References
- Brown, G. D., et al. Hidden killers: human fungal infections. Science Translational Medicine. 4 (165), 113 (2012).
- Brakhage, A. A., Bruns, S., Thywissen, A., Zipfel, P. F., Behnsen, J. Interaction of phagocytes with filamentous fungi. Current Opinion in Microbiology. 13 (4), 409-415 (2010).
- Heinekamp, T., et al. Interference of Aspergillus fumigatus with the immune response. Seminars in Immunopathology. 37 (2), 141-152 (2015).
- Slesiona, S., et al. Persistence versus escape: Aspergillus terreus and Aspergillus fumigatus employ different strategies during interactions with macrophages. PLoS One. 7 (2), 31223 (2012).
- Busetto, S., Trevisan, E., Patriarca, P., Menegazzi, R. A single-step, sensitive flow cytofluorometric assay for the simultaneous assessment of membrane-bound and ingested Candida albicans in phagocytosing neutrophils. Cytometry A. 58 (2), 201-206 (2004).
- Lowe, D. M., et al. A novel assay of antimycobacterial activity and phagocytosis by human neutrophils. Tuberculosis (Edinb). 93 (2), 167-178 (2013).
- Nuutila, J., Lilius, E. M. Flow cytometric quantitative determination of ingestion by phagocytes needs the distinguishing of overlapping populations of binding and ingesting cells. Cytometry A. 65 (2), 93-102 (2005).
- Saresella, M., et al. A rapid evaluation of phagocytosis and killing of Candida albicans by CD13+ leukocytes. Journal of Immunological Methods. 210 (2), 227-234 (1997).
- Fattorossi, A., Nisini, R., Pizzolo, J. G., D’Amelio, R. New, simple flow cytometry technique to discriminate between internalized and membrane-bound particles in phagocytosis. Cytometry. 10 (3), 320-325 (1989).
- Heinzelmann, M., Gardner, S. A., Mercer-Jones, M., Roll, A. J., Polk, H. C. Quantification of phagocytosis in human neutrophils by flow cytometry. Microbiology and Immunology. 43 (6), 505-512 (1999).
- Perticarari, S., Presani, G., Mangiarotti, M. A., Banfi, E. Simultaneous flow cytometric method to measure phagocytosis and oxidative products by neutrophils. Cytometry. 12 (7), 687-693 (1991).
- Sveum, R. J., Chused, T. M., Frank, M. M., Brown, E. J. A quantitative fluorescent method for measurement of bacterial adherence and phagocytosis. Journal of Immunolological Methods. 90 (2), 257-264 (1986).
- de Boer, E. C., Bevers, R. F., Kurth, K. H., Schamhart, D. H. Double fluorescent flow cytometric assessment of bacterial internalization and binding by epithelial cells. Cytometry. 25 (4), 381-387 (1996).
- Hartung, S., et al. Fast and Quantitative Evaluation of Human Leukocyte Interaction with Aspergillus fumigatus Conidia by Flow Cytometry. Cytometry A. 95 (3), 332-338 (2019).
- Fei, C., Lillico, D. M. E., Hall, B., Rieger, A. M., Stafford, J. L. Connected component masking accurately identifies the ratio of phagocytosed and cell-bound particles in individual cells by imaging flow cytometry. Cytometry A. 91 (4), 372-381 (2017).
