A flowcytometric analysis to efficiently quantify multiple innate immune cells and T Cell subsets in human blood

Area of Science:

• Immunology
• Cellular Biology
• Clinical Research

Background:

• Immune cell balance (inflammation vs. immunosuppression) is crucial for cancer, autoimmune, and infectious disease therapies.
• Characterizing immune cell changes in peripheral blood requires efficient flow cytometry protocols using small blood volumes.
• Cryopreserved samples in longitudinal studies can affect flow cytometry results, necessitating robust methods.

Purpose of the Study:

• To establish effective flow cytometry protocols for characterizing immune cell changes in peripheral blood.
• To develop novel 15-color antibody panels for analyzing myeloid and T cell subsets and their functions.
• To compare immune cell profiles in fresh whole blood versus cryopreserved peripheral blood mononuclear cells (PBMCs).

Main Methods:

• Developed two 15-color antibody panels to identify key myeloid and T cell subsets.
• Utilized flow cytometry to analyze cell maturity, stimulatory/inhibitory potential, and migration capabilities.
• Compared immune profiles from fresh whole blood and cryopreserved PBMCs.

Main Results:

• Established reproducible 15-color antibody panels for evaluating immune activation and suppression.
• Successfully characterized myeloid populations (DCs, MO-MDSCs, monocytes) and T cell subsets.
• Demonstrated protocol utility for both fresh whole blood and cryopreserved PBMCs.

Conclusions:

• The novel antibody panels enable efficient evaluation of immune activation and suppression balance.
• The presented gating strategies support specific myeloid and T cell subset identification.
• The protocol is reproducible and applicable to both fresh and cryopreserved blood samples for clinical studies.