A novel real time imaging platform to quantify macrophage phagocytosis.
A novel real time imaging platform to quantify macrophage phagocytosis.
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DOI：
10.1016/j.bcp.2016.07.011
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发表时间：
2016-09-15
影响因子：
5.8
通讯作者：
Greaves, David R.
中科院分区：
文献类型：
--
作者：
Kapellos, Theodore S.;Taylor, Lewis;Lee, Heyne;Cowley, Sally A.;James, William S.;Iqbal, Asif J.;Greaves, David R.
病原体、凋亡细胞和碎片的吞噬作用是巨噬细胞在宿主防御和组织稳态中功能的关键特征。体外巨噬细胞吞噬作用的定量传统上具有技术挑战性。在这里，我们报告了IncuCyte ZOOM®真实的实时成像平台的优化和验证，该平台用于基于pHrodo®病原体生物颗粒的巨噬细胞吞噬作用，该病原体生物颗粒仅在位于吞噬溶酶体的酸性环境中时发出荧光。使用自动化IncuCyte™ Basic软件进行图像分析和荧光定量。生物颗粒数的滴定表明，该系统比荧光分光光度计更灵敏，因为它可以在使用20倍以下的E.大肠杆菌生物颗粒。我们通过研究小鼠肺泡、骨髓和腹腔巨噬细胞的吞噬作用来举例说明这种真实的时间成像平台的能力。我们进一步证明了这个平台的能力，以研究调节的吞噬过程中，作为药理学抑制剂的吞噬作用抑制生物颗粒的摄取浓度依赖性的方式，而调理素增强吞噬作用。我们还研究了巨噬细胞极化对E.大肠杆菌吞噬作用。骨髓源性巨噬细胞（BMDM）与M2刺激，如IL-4和IL-10引发导致更高的吞噬的生物颗粒相比，M1极化。此外，我们证明了脂多糖（LPS）对BMDM的耐受性导致E。大肠杆菌生物颗粒吞噬作用。这种新颖的真实的时间测定将使研究人员能够以更高的准确度和灵敏度量化巨噬细胞吞噬作用，并将允许在体外调查有限的初级吞噬细胞群体。
Phagocytosis of pathogens, apoptotic cells and debris is a key feature of macrophage function in host defense and tissue homeostasis. Quantification of macrophage phagocytosis in vitro has traditionally been technically challenging. Here we report the optimization and validation of the IncuCyte ZOOM® real time imaging platform for macrophage phagocytosis based on pHrodo® pathogen bioparticles, which only fluoresce when localized in the acidic environment of the phagolysosome. Image analysis and fluorescence quantification were performed with the automated IncuCyte™ Basic Software. Titration of the bioparticle number showed that the system is more sensitive than a spectrofluorometer, as it can detect phagocytosis when using 20× less E. coli bioparticles. We exemplified the power of this real time imaging platform by studying phagocytosis of murine alveolar, bone marrow and peritoneal macrophages. We further demonstrate the ability of this platform to study modulation of the phagocytic process, as pharmacological inhibitors of phagocytosis suppressed bioparticle uptake in a concentration-dependent manner, whereas opsonins augmented phagocytosis. We also investigated the effects of macrophage polarization on E. coli phagocytosis. Bone marrow-derived macrophage (BMDM) priming with M2 stimuli, such as IL-4 and IL-10 resulted in higher engulfment of bioparticles in comparison with M1 polarization. Moreover, we demonstrated that tolerization of BMDMs with lipopolysaccharide (LPS) results in impaired E. coli bioparticle phagocytosis. This novel real time assay will enable researchers to quantify macrophage phagocytosis with a higher degree of accuracy and sensitivity and will allow investigation of limited populations of primary phagocytes in vitro.
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DOI：
10.1038/nrm2867
发表时间：
2010-04
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
通讯作者：
--
DOI：
10.1083/jcb.124.5.677
发表时间：
1994-03
期刊：
The Journal of cell biology
影响因子：
--
作者：
Desjardins M;Huber LA;Parton RG;Griffiths G
通讯作者：
Griffiths G
影响因子：
30.5
作者：
通讯作者：
--
影响因子：
3.7
作者：
KAPLAN, G
通讯作者：
KAPLAN, G
影响因子：
64.8
作者：
Foster, Simmie L.;Hargreaves, Diana C.;Medzhitov, Ruslan
通讯作者：
Medzhitov, Ruslan