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 takes a long time to start 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.
Cellprofiler takes a long time to start manual#
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.
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