Immunocytochemistry-Based Imaging for Visualizing Pediatric Glioma Cell Migration

Begin with a flat-bottom multi-well plate coated with a basement membrane matrix, which contains extracellular polymer and adhesion proteins that mimic the tumor microenvironment's matrix.

Introduce a media containing a neurosphere of glioma cells derived from a pediatric brain tumor, ensuring it is centrally positioned in the well.

The matrix promotes the neurosphere attachment to the well.

Add a background suppressor reagent to minimize non-specific fluorescence.

Next, introduce green fluorophore-conjugated anti-CD44 antibodies.

Transfer the plate to a live-cell imaging system housed within an incubator that maintains the cell's physiological conditions.

Over time, glioma cells express the cell-adhesion protein CD44, which interacts with the adhesion proteins.

This activates the cell signaling pathway and facilitates cell migration from the neurosphere.

The fluorophore-conjugated anti-CD44 antibodies bind to the expressed CD44 on the glioma cells, making them fluoresce.

The appearance of green fluorescent cells moving away from the neurosphere confirms the glioma cell migration.