HCS image with cytoskeletal features shown as an overlay

Optimized, automated assays for HCS

This assay automatically measures changes in cell morphology reflecting changes in the underlying intracellular structure. Specifically, the assay measures the morphology of the whole cell and the nucleus, as well as the number, dimensions, intracellular location, and arrangement of F-actin and microtubule fibers. Cytoskeletal rearrangement is best measured in 20x or 40x magnification using individual channels to track the nucleus, whole cell morphology, F-actin, and microtubules. Additional channels can be used to locate specific protein spots or other cellular features such as nucleoli.

Typical cytoskeletal rearrangement assay

Fixed-cell immunofluorescent assay protocol.

Imaging mode

  • Widefield,
  • 20x or 40x magnification

Automatically measured properties

  • Whole cell shape and dimensions
  • Number of cytoskeletal fibers, their dimensions, and alignment
  • Intracellular arrangement, location, and texture measurements of cytoskeletal labels

Cytoskeletal rearrangement assay images

montage of HCS images and graphical representations of the image data produced

Cytoskeletal rearrangement assay. A549 cells were treated with cytochalasin-D and analyzed using the Thermo Scientific CellInsight CX7 High-Content Analysis Platform. Nuclei are labeled with Invitrogen HCS NuclearMask Blue Stain and F-actin is labeled with Invitrogen Alexa Fluor 488 phalloidin, and the whole cell is demarcated using Invitrogen HCS CellMask Deep Red Stain. The assay automatically segments the cells (yellow overlay) and identifies the location and orientation of actin fibers (green overlay). Actin fibers greater than a threshold length are identified and labeled (red overlay). Individual cells can be identified from bar charts and scatter plots.

Sample experimental data

graph of five different cytoskeletal rearrangement assay parameters

Cellular dose-response to cytochalasin as shown by cell area, nuclear intensity, actin fiber count, tubulin fiber count, and actin fiber intensity.