Real-Time Analysis of Resident T-cell Migration in Different Tumor Regions Using Confocal Microscopy

Begin with a lung tumor slice containing resident T cells.

T cells migrate through the tumor microenvironment and interact with tumor cells and stromal elements, performing immune functions.

The composition of the tumor microenvironment strongly influences T cell displacement and velocity.

Add distinct fluorescently-coupled antibodies to label T cells, tumor cells, and stromal elements in the microenvironment.

Add DAPI, which infiltrates cells with a damaged plasma membrane to stain the nucleus, enabling the visualization of necrotic regions within the tumor.

Observe the stained sample under a confocal microscope.

Using a suitable fluorescence light, select a tumor region of interest containing resident T cells. Evaluate cell viability by assessing DAPI-positive cells.

For real-time analysis, define time intervals and total recording time.

To capture z-stack images, start at a depth below labeled T cells and capture images at various time intervals.

Utilize tracking software to compare the T cell displacement length and average velocity in different tumor regions.