Immunophenotyping of Tissue Samples Using Multicolor Flow Cytometry

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Flow cytometry is a powerful technique for analyzing single cells in suspension.
• Advancements in instrumentation and reagents allow for multi-parameter analysis.
• Characterizing immune cells within complex tissues like tumors remains challenging.

Purpose of the Study:

• To provide a standardized protocol for preparing single-cell suspensions from murine lymphoid and tumor tissues.
• To describe a detailed 17-color polychromatic flow cytometry panel for analyzing tumor-infiltrating leukocytes.
• To facilitate high-dimensional single-cell analysis in cancer immunology research.

Main Methods:

• Development of a stepwise protocol for dissociating murine tissues into single-cell suspensions.
• Design and validation of a 17-color polychromatic flow cytometry panel.
• Application of the protocol and panel to analyze tumor-infiltrating leukocytes.

Main Results:

• Successful preparation of high-quality single-cell suspensions from various murine tissues.
• Demonstration of simultaneous measurement of 17 parameters on single cells.
• Detailed characterization of distinct immune cell populations within the tumor microenvironment.

Conclusions:

• The presented protocol enables robust single-cell suspension preparation for flow cytometry.
• The 17-color panel facilitates comprehensive analysis of tumor-infiltrating leukocytes.
• This methodology supports advanced immunological studies of the tumor microenvironment.