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Introduction

Microscopy-based imaging screens can be applied in a high-throughput format to characterize a broad spectrum of cellular processes. They generate large-data sets and on of the key challenges is the analysis, including identification of cells (with many different shapes) under many different perturbation conditions.

Workflow

Fluorescent markers

Different fluorescent markers are used to identify cells, quantify changes in cell shape and viability, cell cycle, homeostasis as well as biophysical properties and kinetics of particular proteins. A short summary of fluorescent markers is listed below.

DNA stain

DAPI or Hoechst are used for counting cells per well (proliferation/viability) and for cell cycle analysis using plate cytometry or fluorescence microscopy. The cell cycle is analysed by measuring the total emitted fluorescence intensity per nucleus and subsequent gating from the distribution of cells in one well or all wells of a 384-well plate.

Cytoskeleton

Tubulin and actin are visualized using fluorescence-labled antibodies. Cytoskelletal stains are used for image segementation (separation and identification  of single cells in an image or well) and observation of cell morphology (phenotypic profiling).

Cytoplasm

Cytomplasmic stain is achieved by loading the cell with a cell-permeant dye (e.g. AM esters) or by transient or stable expression of recombinant GFP variants. This method is choosen  for normalization purposes in combination with luminescence reporters or for kinetic measurements of cellular processes.

Organelles

Sub-cellular compartments such as endosomes, lipid droplets, etc. are stained using loadeble dyes, antibodies or transfected GFP variants

GFP-fusion proteins and variants

GFP variants fused to target genes enable quantification of kinetics and localisation of highly conserved signaling pathways relevant in a vaiety of diseases.

Most of the mentioned reporters can be combined in one experiment and can serve in multiplexing experiment.

Image analysis

References




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