Detection of alveolar macrophages phagocytosis of fluorescently labeled bacteria by flow cytometry_Chinese Journal of Respiratory and Critical Care Medicine

Authors：

LI Naijian ¹ , PAN Tianhui ² , HE Fang ³ ,  RAN Pixin ¹

1. State Key Laboratory of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510120, P. R. China;
2. The First Clinical College of Guangzhou Medical University, Guangzhou, Guangdong 510120, P. R. China;
3. The School of Basic Medicine, Guangzhou Medical University, Guangzhou, Guangdong 510120, P. R. China;

Corresponding author：

RAN Pixin, Email: pxran@gzhmu.edu.cn

Keywords：

Flow cytometry; Alveolar macrophages; Bacteria; Phagocytosis

DOI：

10.7507/1671-6205.201803034

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Objective To establish a methodology for alveolar macrophages (AMs) phagocytosis of AlexaFluor 488 (AF488) labeled bacteria by flow cytometry.Methods Staphylococcus aureus and Streptococcus pneumoniae were labeled with different concentrations of AF488. A flow cytometric assay was used to quantify in vivo bacterial uptake by AMs. AMs and different ratio of fluorescent-labeled bacteria were incubated at 37 ℃ for 2 hours, 4 hours, 6 hours and 8 hours, respectively. AMs were washed with DPBS and extracellular fluorescence was quenched with 1% (w/v) trypan blue. Trypan blue was aspirated and phagocytosis of fluorescent-labeled bacteria by AMs was measured using a flow cytometry. Confocal microscopy was performed to ensure that bacterial in positive AM had been internalized rather than bound to the cell surface.Results When the concentration of AF488 was more than 50 μg/mL, the labeling rates of Staphylococcus aureus and Streptococcus pneumoniae were higher than 92% (P<0.05), and has quickly reached the upper limit. With the prolongation of incubation time, the phagocytic rate of AMs increased from 20.4% at 2 hours to 76.5% at 8 hours. With the increase in the number of bacteria, the phagocytic rate of AMs increased from 7.7% by ratio of 1∶10 to 85.1% by ratio of 1∶300.Conclusion Detection of AMs phagocytosis of AF488 labeled bacteria by flow cytometry is an effective method, but the dye concentration, incubation time and the proportion of bacteria will influence the results.

Citation： LI Naijian, PAN Tianhui, HE Fang, RAN Pixin. Detection of alveolar macrophages phagocytosis of fluorescently labeled bacteria by flow cytometry. Chinese Journal of Respiratory and Critical Care Medicine, 2019, 18(5): 479-483. doi: 10.7507/1671-6205.201803034 Copy

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3.	Taylor AE, Finney-Hayward TK, Quint JK, et al. Defective macrophage phagocytosis of bacteria in COPD. Eur Respir J, 2010, 35(5): 1039-1047.
4.	Fitzpatrick AM, Holguin F, Teague WG, et al. Alveolar macrophage phagocytosis is impaired in children with poorly controlled asthma. J Allergy Clin Immunol, 2008, 121(6): 1372-1378.
5.	Steinkamp JA, Wilson JS, Saunders GC, et al. Phagocytosis: flow cytometric quantitation with fluorescent microspheres. Science, 1982, 215(4528): 64-66.
6.	Pitz AM, Perry GA, Jensen-Smith HCA, et al. A flow cytometric assay to quantify in vivo bacterial uptake by alveolar macrophages. J Microbiol Methods, 2010, 81(2): 194-196.
7.	Inoue M, Ishibashi Y, Nogawa H, et al. Carbocisteine promotes phagocytosis of apoptotic cells by alveolar macrophages. Eur J Pharmacol, 2012, 677(1-3): 173-179.
8.	金齐力, 姜丽娜, 姚春艳, 等. 流式细胞术检测单核巨噬细胞吞噬荧光素标记结核分枝杆菌的方法学探讨. 蚌埠医学院学报, 2008, 33(5): 505-508.
9.	王爱清, 万建美, 彭丽, 等. 流式细胞术检测大鼠肺泡巨噬细胞吞噬功能方法探讨. 激光杂志, 2014, 35(1): 39-41.
10.	李煜, 齐丽娟, 迈一冰, 等. 比较流式细胞术和鸡红细胞法检测小鼠腹腔巨噬细胞吞噬功能. 毒理学杂志, 2012, 26(2): 133-135.
11.	Kendall CG, Stockton AM, Leicht S, et al. Amine analysis using AlexaFluor 488 succinimidyl ester and capillary electrophoresis with laser-induced fluorescence. J Anal Methods Chem, 2015, 2015: 368362.
12.	Lindhoud S, Westphal AH, Visser AJ, et al. Fluorescence of Alexa Fluor dye tracks protein folding. PLoS One, 2012, 7(10): e46838.
13.	陈亚东, 陆罗定, 俞萍, 等. 流式细胞术检测小鼠腹腔巨噬细胞吞噬能力的方法学探讨. 中国免疫学杂志, 2014, 8(30): 1074-1077.

1. Donnelly LE, Barnes PJ. Defective phagocytosis in airways disease. Chest, 2012, 141(4): 1055-1062.
2. Berenson CS, Garlipp MA, Grove LJ, et al. Impaired phagocytosis of nontypeable haemophilus influenzae by human alveolar macrophages in chronic obstructive pulmonary disease. J Infect Dis, 2006, 194(10): 1375-1384.
3. Taylor AE, Finney-Hayward TK, Quint JK, et al. Defective macrophage phagocytosis of bacteria in COPD. Eur Respir J, 2010, 35(5): 1039-1047.
4. Fitzpatrick AM, Holguin F, Teague WG, et al. Alveolar macrophage phagocytosis is impaired in children with poorly controlled asthma. J Allergy Clin Immunol, 2008, 121(6): 1372-1378.
5. Steinkamp JA, Wilson JS, Saunders GC, et al. Phagocytosis: flow cytometric quantitation with fluorescent microspheres. Science, 1982, 215(4528): 64-66.
6. Pitz AM, Perry GA, Jensen-Smith HCA, et al. A flow cytometric assay to quantify in vivo bacterial uptake by alveolar macrophages. J Microbiol Methods, 2010, 81(2): 194-196.
7. Inoue M, Ishibashi Y, Nogawa H, et al. Carbocisteine promotes phagocytosis of apoptotic cells by alveolar macrophages. Eur J Pharmacol, 2012, 677(1-3): 173-179.
8. 金齐力, 姜丽娜, 姚春艳, 等. 流式细胞术检测单核巨噬细胞吞噬荧光素标记结核分枝杆菌的方法学探讨. 蚌埠医学院学报, 2008, 33(5): 505-508.
9. 王爱清, 万建美, 彭丽, 等. 流式细胞术检测大鼠肺泡巨噬细胞吞噬功能方法探讨. 激光杂志, 2014, 35(1): 39-41.
10. 李煜, 齐丽娟, 迈一冰, 等. 比较流式细胞术和鸡红细胞法检测小鼠腹腔巨噬细胞吞噬功能. 毒理学杂志, 2012, 26(2): 133-135.
11. Kendall CG, Stockton AM, Leicht S, et al. Amine analysis using AlexaFluor 488 succinimidyl ester and capillary electrophoresis with laser-induced fluorescence. J Anal Methods Chem, 2015, 2015: 368362.
12. Lindhoud S, Westphal AH, Visser AJ, et al. Fluorescence of Alexa Fluor dye tracks protein folding. PLoS One, 2012, 7(10): e46838.
13. 陈亚东, 陆罗定, 俞萍, 等. 流式细胞术检测小鼠腹腔巨噬细胞吞噬能力的方法学探讨. 中国免疫学杂志, 2014, 8(30): 1074-1077.

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Detection of alveolar macrophages phagocytosis of fluorescently labeled bacteria by flow cytometry

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