HCS image with autophagy features shown as an overlay

Optimized, automated assays for HCS

Autophagy is characterized by the recruitment of LC3B, a ubiquitin-like protein, from the cytosol and its subsequent association with the phagophore. This localization results in discrete puncta as the protein is incorporated into the lipid layers of the vesicles, and it serves as a general marker for autophagic membranes and for monitoring the autophagic process as it develops. In this assay, quantitation of LC3B protein on autophagic vesicles is achieved using a fixed end-point assay based on immunofluorescence detection. Cells are identified using a nuclear stain and LC3B expression associated with each cell is measured. Additional channels can be used to monitor other cellular processes.

Typical autophagy assay

Fixed-cell immunofluorescence–assay protocol

Imaging mode

  • Widefield
  • 20x magnification

Automatically measured properties

  • Fluorescence intensity, morphology, and count values for each object
  • Fluorescence intensity, morphology, and count values of spots in different cell regions
  • Average fluorescence intensity difference and ratios between different regions for each cell
  • Intensity and count ratio between channels within different regions for each cell

Autophagy assay images

Two-panel HCS autophagy assay image showing stained cells and software overlay

A549 cells treated to induce autophagy and imaged. (Left) A549 cells were treated with chloroquine and stained with Invitrogen Hoechst 33342, Invitrogen HCS CellMask Deep Red, and an anti-LC3B with Invitrogen Alexa Fluor 488 goat anti-rabbit antibody. Cells accumulate LC3B at higher chloroquine concentrations. (Right) Using Thermo Scientific CellInsight CX5 high-content platform and Thermo Scientific HCS Studio Software, autophagy features were automatically identified: nuclei (blue), cells (green boundary), and LC3B was assayed both by granule count (pink) and by measuring fluorescence intensity in the 488 channel.

Sample experimental data

graph of granules vs chloroquine concentration

Dose-response plot of LC3B granules per cell with increasing chloroquine concentration.

graph of fluorescence vs chloroquine concentration

Dose-response plot of LC3B fluorescence intensity with increasing chloroquine concentration.