HCS image with stem cell markers shown as an overlayOptimized, automated assays for HCS

This automated assay monitors the transition of pluripotent stem cells (PSC) to a mesodermal and then committed cardiomyocyte phenotype using a fixed-cell imaging protocol. In a 3-channel assay, cells are fixed and stained with a nuclear stain for localization and with primary antibodies against nuclear transcription factors associated with pluripotency (Oct4) and cardiac differentiation (Nkx2.5). The primary antibodies are detected with Invitrogen Alexa Fluor dye–conjugated secondary antibodies at appropriate wavelengths for subsequent multiplexing. The nuclear stain provides a mask for automatic compartmental analysis. Oct4 and Nkx2.5 nuclei are then enumerated based on detecting the secondary label within the total nuclear mask, to yield a labeling index for each marker.

Typical stem cell differentiation assay

Fixed-cell immunofluorescent assay protocol.

Imaging mode

  • Widefield
  • 10x magnification

Automatically measured properties

  • Nuclear mask identification
  • Spots identified and localized by channel

Stem cell differentiation assay images

Immunofluorescent detection of Oct4 (green) and NKx2.5 (red) to determine differentiation status in H9 cells stained with DAPI (blue).

Identifying differentiated stem cells. DAPI staining is used to automatically generate a nuclear mask (blue outline), then cells positive for the differentiation marker (in this case Oct4) within the nuclear region are highlighted in red.


Sample experimental data

Results from stem cell differentiation assay. The percentages of Oct4+ and Nkx2.5+ nuclei were quantified in cells fixed at the indicated time points. Prior to differentiation (Day 0), nearly 100% of all cells were Oct4+/Nkx2.5–, consistent with a pluripotent state. At Day 6, greater than 90% of cells analyzed were co-positive for both markers. After day 6, the frequency of Oct4+ cells declined, consistent with a loss of pluripotency and a transition to a terminally differentiated cardiomyocyte phenotype.