Autophagy profiling in single cells with open source CellProfiler-based image analysis

Single cell-based analysis of macroautophagy/autophagy is largely achieved through the use of fluorescence microscopy to detect autophagy-related proteins that associate with autophagic membranes and therefore can be quantified as fluorescent puncta. In this context, an automated analysis of the number and size of recognized puncta is preferable to a manual count, because more reliable results can be generated in a short time. Here we present a method for open source CellProfiler software-based analysis for quantitative autophagy assessments using GFP-tagged WIPI1 (WD repeat domain, phosphoinositide interacting 1) images acquired with Airyscan or confocal laser-scanning microscopy. The CellProfiler protocol is provided as a ready-to-use software pipeline, and the creation of this pipeline is detailed in both text and video formats. In addition, we provide CellProfiler pipelines for endogenous SQSTM1/p62 (sequestosome 1) or intracellular lipid droplet (LD) analysis, suitable to assess forms of selective autophagy. All protocols and software pipelines can be quickly and easily adapted for the use of alternative autophagy markers or cell types, and can also be used for high-throughput purposes.<b>Abbreviations:</b> AF Alexa Fluor ATG autophagy related BafA1 bafilomycin A1 BSA bovine serum albumin DAPI 4,6-diamidino-2-phenylindole DMEM Dulbecco’s modified Eagle’s medium DMSO dimethyl sulfoxide EDTA ethylenediaminetetraacetic acid EBSS Earle’s balanced salt solution FBS fetal bovine serum GFP green fluorescent protein LD lipid droplet LSM laser scanning microscope MAP1LC3B microtubule associated protein 1 light chain 3 beta MTOR mechanistic target of rapamycin kinase PBS phosphate-buffered saline PIK3C3/VPS34 phosphatidylinositol 3-kinase catalytic subunit type 3 SQSTM1 sequestosome 1 TIFF tagged image file format U2OS U-2 OS cell line WIPI WD repeat domain, phosphoinositide interacting

基于单细胞的巨自噬（macroautophagy，又称自噬）分析目前主要通过荧光显微镜检测结合自噬膜的自噬相关蛋白，并以荧光斑点的形式进行定量分析。在此场景下，对识别出的斑点数量与尺寸进行自动化分析，相较于人工计数更具优势，因其可在短时间内获得更可靠的结果。本文报道一种基于开源CellProfiler软件的分析方法，用于定量评估自噬水平，其所用图像为通过Airyscan或共聚焦激光扫描显微镜获取的、绿色荧光蛋白（green fluorescent protein, GFP）标记的WD重复域磷酸肌醇相互作用蛋白1（WD repeat domain, phosphoinositide interacting 1, WIPI1）图像。本研究提供的CellProfiler分析方案以可直接使用的软件工作流形式呈现，其构建过程同时以文本与视频格式进行了详细说明。此外，本研究还提供了用于内源性SQSTM1/p62（sequestosome 1，又称隔离体1）或细胞内脂滴（lipid droplet, LD）分析的CellProfiler工作流，适用于评估选择性自噬的不同形式。所有方案与软件工作流均可快速便捷地适配其他自噬标志物或细胞系的相关分析，同时也可用于高通量检测场景。

**缩略词：**
AF：Alexa Fluor
ATG：autophagy related（自噬相关）
BafA1：巴弗洛霉素A1（bafilomycin A1）
BSA：牛血清白蛋白（bovine serum albumin）
DAPI：4',6-二脒基-2-苯基吲哚（4,6-diamidino-2-phenylindole）
DMEM：达尔伯克改良伊格尔培养基（Dulbecco’s modified Eagle’s medium）
DMSO：二甲基亚砜（dimethyl sulfoxide）
EDTA：乙二胺四乙酸（ethylenediaminetetraacetic acid）
EBSS：厄尔平衡盐溶液（Earle’s balanced salt solution）
FBS：胎牛血清（fetal bovine serum）
GFP：绿色荧光蛋白（green fluorescent protein）
LD：脂滴（lipid droplet）
LSM：激光扫描显微镜（laser scanning microscope）
MAP1LC3B：微管相关蛋白1轻链3β（microtubule associated protein 1 light chain 3 beta）
MTOR：雷帕霉素机制靶蛋白激酶（mechanistic target of rapamycin kinase）
PBS：磷酸盐缓冲液（phosphate-buffered saline）
PIK3C3/VPS34：磷脂酰肌醇3-激酶催化亚基3型（phosphatidylinositol 3-kinase catalytic subunit type 3）
SQSTM1：隔离体1（sequestosome 1，又称p62）
TIFF：标记图像文件格式（tagged image file format）
U2OS：U-2 OS细胞系（U-2 OS cell line）
WIPI：WD重复域磷酸肌醇相互作用蛋白（WD repeat domain, phosphoinositide interacting）