Department of Pharmacology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China.
Translational Medicine Research Center, People's Hospital Of Zhengzhou, Zhengzhou, Henan, China.
BMC Neurosci. 2022 Mar 31;23(1):21. doi: 10.1186/s12868-022-00703-1.
Microglia, the resident immune cells in the central nervous system, accrue autofluorescent granules inside their cytoplasm throughout their lifespan. In this report, we studied the impacts of autofluorescence on widely used fluorescence-based techniques to study microglia, including flow cytometry, immunofluorescence staining, and live imaging.
The failed attempt of using fluorescein isothiocyanate (FITC) conjugated antibody to detect lymphocyte-activation gene 3 protein in microglia prompted us to compare the sensitivity of FITC, phycoerythrin (PE) and allophycocyanin (APC) conjugated antibodies to detect surface protein expression in microglia. We found that PE outperformed FITC and APC as the fluorophore conjugated to antibody for flow cytometry by overcoming the interference from microglia autofluorescence. To identify the location and source of microglia autofluorescence, we did confocal imaging and spectral analysis of microglia autofluorescence on fixed brain tissues, revealing that microglia autofluorescence emitted from cytoplasmic granules and displayed a multi-peak emission spectrum. We recommended removing autofluorescence by lipofuscin removing agents when staining intracellular proteins in microglia with the immunofluorescence techniques. On live brain slices, autofluorescent granules reduced the amplitudes of calcium signals in microglial somata derived from GCaMP6s fluorescence and thus needed to be excluded when selecting regions of interest (ROI).
In conclusion, autofluorescence is a critical factor to consider when designing experiments and interpreting results based on fluorescence-based techniques to study microglia.
小胶质细胞是中枢神经系统中的固有免疫细胞，在其整个生命周期中，其细胞质内会积累自发荧光颗粒。在本报告中，我们研究了自发荧光对广泛用于研究小胶质细胞的荧光基技术的影响，包括流式细胞术、免疫荧光染色和活体成像。
使用异硫氰酸荧光素（FITC）缀合抗体检测小胶质细胞中的淋巴细胞激活基因 3 蛋白的尝试失败促使我们比较了 FITC、藻红蛋白（PE）和别藻青蛋白（APC）缀合抗体检测小胶质细胞表面蛋白表达的灵敏度。我们发现，PE 作为抗体缀合的荧光团优于 FITC 和 APC，可用于流式细胞术，克服了小胶质细胞自发荧光的干扰。为了确定小胶质细胞自发荧光的位置和来源，我们对固定脑组织中的小胶质细胞自发荧光进行了共聚焦成像和光谱分析，结果表明小胶质细胞自发荧光来自细胞质颗粒，并显示出多峰发射光谱。我们建议在用免疫荧光技术染色小胶质细胞内蛋白时，使用脂褐素去除剂去除自发荧光。在活体脑切片上，自发荧光颗粒降低了源自 GCaMP6s 荧光的小胶质细胞体钙信号的幅度，因此在选择感兴趣区域（ROI）时需要将其排除。
总之，在设计实验和基于荧光基技术解释小胶质细胞研究结果时，自发荧光是一个需要考虑的关键因素。
