Optimized protocol for immunophenotyping of melanoma and tumor-bearing skin from mouse

Area of Science:

• Immunology
• Cell Biology
• Dermatology

Background:

• Isolating immune cells from solid tissues like skin presents significant challenges compared to blood or lymphoid organs.
• Existing methods often result in low cell viability or biased cell representation, hindering comprehensive immune profiling.
• Skin-resident immune cells play crucial roles in local immunity and disease pathogenesis.

Purpose of the Study:

• To develop and validate a robust protocol for the enzymatic digestion of skin tissue.
• To optimize conditions for preserving immune cell viability and function during the isolation process.
• To enable the sensitive detection of low-abundance immune cell populations within the skin, specifically in a cancer model.

Main Methods:

• A step-by-step protocol for enzymatic skin digestion was established using a murine melanoma model.
• Optimization of digestion time, temperature, and enzyme concentrations was performed.
• Flow cytometry was employed to assess immune cell viability and enumerate specific immune cell subsets, including group 2 innate lymphoid cells.

Main Results:

• The developed protocol effectively dissociates murine skin, yielding high numbers of viable immune cells.
• The method demonstrated superior performance in preserving cell integrity compared to standard dissociation techniques.
• Low-abundant immune cell populations, including group 2 innate lymphoid cells, were successfully detected and characterized in skin digests.

Conclusions:

• This protocol provides a reliable method for isolating viable immune cells from murine skin, particularly within a melanoma microenvironment.
• The technique facilitates the study of cellular immunity in skin-associated conditions and cancer research.
• It opens avenues for deeper investigation into the role of rare immune cells in skin health and disease.