The automated or manual counting of, for example, GFP-WIPI1 puncta in single cells is one of the recommended methods for the quantitative analysis of autophagy. Due to their phosphatidylinositol-3-phosphate dependent binding to autophagic membranes, endogenous or GFP-tagged overexpressed WIPI β-propellers are used to make autophagic membranes visible and appear as fluorescent puncta under the light microscope. There are four human WIPI β-propellers (WIPI1, WIPI2, WDR45B/WIPI3, and WDR45/WIPI4) that function as phosphatidylinositol-3-phosphate effectors at the nascent autophagosome. We have established a fluorescence-based autophagy assay using human WIPI (WD repeat domain, phosphoinositide interacting) β-propellers that we previously identified. In addition, the detection of autophagy receptors, such as SQSTM1, or the specific cargo itself that is recognized by autophagy receptors, such as lipid droplets (LDs) during lipophagy, is used to assess forms of selective autophagy. To achieve this goal, endogenous ATG proteins are detected by indirect immunofluorescence using fixed cells, or alternatively, overexpressed ATG protein fusions with fluorescent proteins such as the green fluorescent protein (GFP) are used to detect autophagic membranes in both fixed and living cells or tissues. ĭepending on the stage of the autophagy pathway, autophagic membranes are decorated with specific combinations of autophagy related (ATG) proteins that can be used to identify autophagic membranes in single cells. In this context, fluorescence-based detection of autophagic membranes and the determination of their abundance is one of the most widely used method for assessing autophagy. Therefore, it is recommended to use a variety of different autophagy assessments that qualify for robust quantifications. Due to the fact that the autophagosome is formed de novo and quickly comes into contact with the lysosomal compartment, the dynamics of this process make its analysis difficult, especially in human diseases models characterized by improper autophagy. Macroautophagy (hereafter referred to as autophagy) is a lysosomal pathway of degradation and is characterized by the formation of a double-membrane vesicle, called an autophagosome, that sequesters cytoplasmic material in a stochastic or selective manner.
Abbreviations: 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
#Cellprofiler saving images software
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.
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. 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. 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. 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. 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.
0 kommentar(er)
