20.500.12556/RUL-171715 Razvoj in optimizacija nabora označevalcev za karakterizacijo in fenotipizacijo celic gama delta T s pretočno citometrijo Development and optimization of a comprehensive spectral flow cytometry panel for characterization and phenotyping of gamma delta T cells Celice gama delta (γδ) T pri zdravih posameznikih predstavljajo med 1 in 5 % celic T, vendar se njihova pogostost in sestava podskupin lahko izrazito spremenita ob različnih bolezenskih stanjih. Namen magistrske naloge je bil vzpostaviti in optimizirati protokol barvanja ter analize na spektralnem pretočnem citometru za večbarvno fenotipizacijo imunskih celic, s poudarkom na celicah γδ T in njihovih podskupinah (Vδ1, Vδ2, Vγ9). Identifikacijo izbranih populacij smo izvedli z uporabo označevalcev za specifične celične markerje na mononuklearnih celicah periferne krvi (MCPK) in analizo s spektralnim pretočnim citometrom. V treh sklopih poskusov smo optimizirali barvanje z barvilom za določanje živosti celic, površinsko barvanje s preliminarnim naborom 18 fluoroforjev ter razširjeno površinsko in znotrajcelično barvanje z naborom 30 fluoroforjev. Optimizirali smo tudi postopek izolacije MCPK iz dveh vrst začetnega materiala: levkocitno-trombocitne plasti in polne heparinizirane periferne krvi, pri čemer smo uporabili vzorce treh zdravih odraslih darovalcev za vsako vrsto materiala. Hkrati smo primerjali tudi štiri različne pristope shranjevanja celic pred barvanjem. Razviti razširjeni nabor 30 fluoroforjev predstavlja osnovo za nadaljnjo uporabo pri analizi relevantnih kliničnih vzorcev. 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. celice gama delta T pretočna citometrija nabor označevalcev fenotipizacija karakterizacija gamma delta T cells flow cytometry flow cytometry panel phenotyping characterization true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-08-31 07:17:15 2025-08-31 07:17:18 2025-09-17 03:50:19 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2026-08-29 261893 247550979 50791.pdf 50791.pdf 1 27DF86E4C17F67028F0165E8AD162629 78e19c606a8fb6fc6e653d884493b7211baa357de5c8595129d27a3420e36fac a01363d5-8629-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=215097 Biotehniška fakulteta 0 0 0