Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular Virology of MOE/MOH, Fudan University, Shanghai, 201508, China.
TB Center, Shanghai Emerging and Re-emerging Institute, Shanghai, 201508, China.
Sci Rep. 2021 Jan 13;11(1):1096. doi: 10.1038/s41598-020-79425-5.
Immunofluorescence is indispensable to monitor redistribution of proteins involved in phagosome-lysosome association pathway-relevant (P-LApr) proteins. The software digitizing the signals of these proteins in an unbiased and automated manner is generally costly and not widely available. The open-source ImageJ plugin EzColocalization, which is for co-localization analysis of reporters in cells, was not straightforward and sufficient for such analysis. We describe here the input of custom Java code in a novel tailored protocol using EzColocalization to digitize the signals of punctum-distributed P-LApr proteins co-localized with phagosomes and to calculate percentages of phagosomes engaged. We showed that SYBR Gold nucleic acid dye could visualize intracellular mycobacteria that did not express a fluorescent protein. This protocol was validated by showing that IFN-γ enhanced the co-localization of a punctum-distributed P-LApr protein (LC3) with Mycobacterium bovis BCG in the monocyte/macrophage-like RAW264.7 cells and that there was greater co-localization of LC3 with BCG than with M. tuberculosis H37Rv in bone marrow-derived macrophages (BMDMs). Although BCG and a derived strain (rBCG-PA) showed a similarly high degree co-localization with LC3 in BMDMs, in RAW264.7 cells BCG showed much less co-localization with LC3 than rBCG-PA indicating the need for caution in interpreting biological significance from studies in cell lines.
免疫荧光对于监测参与吞噬体-溶酶体相关途径的蛋白质（P-LApr 蛋白）重分布是不可或缺的。以无偏倚和自动化的方式对这些蛋白质的信号进行数字化的软件通常成本高昂且无法广泛获得。用于细胞内报告蛋白共定位分析的开源 ImageJ 插件 EzColocalization 并不直接适用于此类分析。我们在这里描述了一种新的定制协议，使用 EzColocalization 输入自定义 Java 代码，以数字化与吞噬体共定位的点状分布的 P-LApr 蛋白的信号，并计算参与吞噬体的百分比。我们表明，SYBR Gold 核酸染料可以可视化不表达荧光蛋白的细胞内分枝杆菌。该方案通过显示 IFN-γ增强单核细胞/巨噬细胞样 RAW264.7 细胞中点状分布的 P-LApr 蛋白（LC3）与牛分枝杆菌 BCG 的共定位，以及 LC3 与 BCG 的共定位比与结核分枝杆菌 H37Rv 更密切，得到了验证。虽然 BCG 和衍生株（rBCG-PA）在骨髓来源的巨噬细胞（BMDMs）中与 LC3 显示出高度共定位，但在 RAW264.7 细胞中，BCG 与 LC3 的共定位程度远低于 rBCG-PA，这表明在细胞系研究中解释生物学意义时需要谨慎。
