Gamma delta (γδ) T cells represent between 1 and 5 % of total T cells in healthy individuals, but their frequency and subset composition can change significantly under various pathological conditions. The aim of this master's thesis was to establish and optimize a staining and analysis protocol on a spectral flow cytometer for multicolor phenotyping of immune cells, with a particular focus on γδ T cells and their subsets (Vδ1, Vδ2, Vγ9). Selected immune cell populations were identified using markers for specific cell surface and intracellular proteins on peripheral blood mononuclear cells (PBMCs), followed by spectral flow cytometry analysis. In three experimental phases, we optimized staining with a cell viability dye, surface staining using a preliminary panel of 18 fluorophores, and combined surface and intracellular staining using an extended panel of 30 fluorophores. Additionally, we refined the isolation protocol for PBMCs obtained from two types of starting material: buffy coat and whole heparinized peripheral blood, each collected from three healthy adult donors. We also compared four different approaches to cell storage prior to staining. The developed expanded panel of 30 fluorochromes provides a foundation for use in the analysis of relevant clinical samples.
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