Protocol for profiling circulating T cell subsets, their homing and regulatory markers, in pediatric samples using a...

Area of Science:

• Immunology
• Cell Biology
• Hematology

Background:

• Characterizing T cell subsets is crucial for understanding immune responses.
• Efficiently analyzing limited pediatric blood samples presents a challenge.
• Multiparameter flow cytometry is a powerful tool for immune cell profiling.

Purpose of the Study:

• To present a detailed protocol for profiling T cell subsets and immune markers.
• To optimize the analysis of human peripheral blood mononuclear cells (PBMCs).
• To enable comprehensive analysis using minimal pediatric blood volumes.

Main Methods:

• Isolation, cryopreservation, thawing, and stimulation of human PBMCs.
• Multiparameter flow cytometry for analyzing T cell phenotypes.
• Stimulation of cells with staphylococcal enterotoxin B (SEB), lipopolysaccharide (LPS), or zymosan (ZYM).

Main Results:

• A comprehensive protocol for T cell subset and marker profiling is established.
• The protocol allows for the analysis of unstimulated or stimulated PBMCs.
• Efficient utilization of minimal pediatric blood volumes for valuable sample analysis.

Conclusions:

• This protocol provides a robust method for T cell subset and phenotype analysis.
• The described flow cytometry approach maximizes information from limited blood samples.
• The protocol is applicable to both unstimulated and stimulated immune cell analyses.