Optimized, automated assays for HCS

Endocytosis is involved in many cellular processes such as nutrient acquisition and receptor signaling. Analysis of the extent of internalization of a given molecule can be used as a marker of endocytosis. Commonly used markers include ligands such as LDL, EGF, or transferrin as well as fluid phase markers, for example 10,000 MW dextrans conjugated to fluorophores to determine their localization.

The pHrodo family of pH-sensitive dyes is particularly useful for tracking internalization. pHrodo dyes are almost non-fluorescent at neutral pH and fluoresce brightly in acidic environments. Conjugated to an appropriate ligand, these probes make excellent no-wash assay substrates since they are only detected once they are internalized into the acidic endosomal pathway. 

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Typical endocytosis assay

Live or fixed-cell assay protocol.

Imaging mode

  • Widefield or confocal
  • 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

Endocytosis assay images

U2OS cells stably expressing a GFP-EGF receptor construct and incubated with pHrodo red EGF conjugate were treated with PitStop 2 inhibitor (Abcam, Inc.) to inhibit endocytosis. Cells were stained with Invitrogen Hoechst 33342, and pHrodo red EGF conjugate. Stained cells were imaged using Thermo Scientific CellInsight CX5 high-content platform (row A) and analyzed using HCS Studio Software. Internalized EGF receptors and the EGF ligand were automatically identified (row B): nuclei (blue), cells (green boundary), and EGF or the EGF receptor were assayed both by granule count (red for pHrodo EGF or green for GFP-EGF receptor).


Sample experimental data

Dose-dependent loss of EGF receptor (green trace) and EGF internalization (red trace) with increasing concentrations of Pitstop 2 inhibitor (Abcam, Inc.) in U2OS cells. At high concentrations of Pitstop 2 inhibitor, cells are unable to internalize the EGF receptor and its ligand.

Resources

Instrument systems

BioProbes Journal article

HCS reagents optimized for use with your HCS platform