20.500.12556/RUL-184913 Vzpostavitev večparametrične pretočne citometrije za analizo imunosupresivnih lastnosti regulatornih celic T Establishment of multiparametric flow cytometry for the analysis of immunosuppressive properties of regulatory T cells Regulatorne celice T so podskupina CD4+ T celic, karakterizirane z izražanjem FOXP3 transkripcijskega faktorja in visokim izražanjem CD25. So ključne za ohranjanje imunske homeostaze saj zavirajo delovanje konvencionalnih efektorskih celic T, in preprečujejo pretirane imunske odzive in razvoj avtoimunosti. Namen magistrske naloge je bil izvesti in vitro supresijski test ter vzpostaviti večparametrični sistem na osnovi spektralne pretočne citometrije za raziskavo imunosupresivnih kapacitet regulatornih celic T. Regulatorne in konvencionalne celice T smo pridobili iz mišjih vranic z optimiziranim postopkom izolacije. Celice smo aktivirali s pomočjo aktivacijskih kroglic ali koktejla za stimulacijo celic. Kontrolna skupina vzorcev je ostala nestimulirana. Med seboj smo primerjali aktivacijo in proliferacijo konvencionalnih celic T v odsotnosti ali ob prisotnosti regulatornih celic T. Preko barvanja celičnih markerjev, ki kažejo na aktivacijo celic, in barvanjem z barvilom CFSE za spremljanje proliferacije, smo s pomočjo spektralne citmetrije ovrednotili imunosupresivno kapaciteto regulatornih celic T v ko-kulturi. Prisotnost regulatornih celic T v vzorcu celic, aktiviranih s kroglicami, statistično značilno zniža izražanje aktivacijskih markerjev - CD25 in CD69. Prisotnost regulatornih celic T vpliva tudi na parametre modeliranja proliferacije - število nedeljenih celic in replikacijski indeks pri vzorcih aktiviranih z aktivacijskimi kroglicami ter odstotek deljenih celic pri vzorcih aktiviranih s koktejlom za stimulacijo celic. In vitro imunosupresijski test predstavlja prvi korak pri preučevanju supresivne kapacitete regulatornih celic T. Znanje pridobljeno s tem testom služi kot osnova za nadaljnje in vivo študije hkrati pa omogoča prenos metodologije tudi na druge celične sisteme, kot so humane ali gensko modificirane celice. Regulatory T cells are a subset of CD4+ T cells characterized by the expression of the transcription factor FOXP3 and high expression of CD25. They play a crucial role in maintaining immune homeostasis by suppressing the activity of conventional effector T cells, thereby preventing excessive immune responses and the development of autoimmunity. The aim of this master's thesis was to perform an in vitro suppression assay and establish a multiparametric system based on spectral flow cytometry for the investigation of the immunosuppressive capacities of regulatory T cells. Regulatory and conventional T cells were isolated from murine spleens using an optimized isolation protocol. The cells were activated either with activation beads or with a cell stimulation cocktail, while the control group remained unstimulated. The activation and proliferation of conventional T cells were compared in the absence and presence of regulatory T cells. By staining cellular markers indicative of cell activation and using the CFSE dye to monitor proliferation, the immunosuppressive capacity of regulatory T cells in co-culture was evaluated using spectral flow cytometry. The presence of regulatory T cells in samples activated with beads significantly reduced the expression of the activation markers CD25 and CD69 and affected proliferation modeling parameters, including the number of undivided cells and the replication index in bead-activated samples, as well as the percentage of divided cells in samples activated with the cell stimulation cocktail. The in vitro immunosuppression assay represents the first step in studying the suppressive capacity of regulatory T cells. The knowledge gained from this assay serves as a foundation for subsequent in vivo studies while also enabling the transfer of the methodology to other cellular systems, such as human or genetically modified cells. regulatorne celice T konvencionalne celice T in vitro test imunosupresije spektralna pretočna citometrija nabor označevalcev proliferacija regulatory T cells conventional T cells in vitro immunosuppression assay spectral flow cytometry flow cytometry panel proliferation true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-07-17 07:16:54 2026-07-17 07:17:00 2026-07-18 04:19:00 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2027-07-15 282169 285213187 58088.pdf 58088.pdf 1 D8A374C02134347FAE6877801D7F672C fdcefeb98360fd2f0675e51bd8a096a10f6ad82b296ac990222995ab48fc3103 89cf8ef8-819e-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=240309 Biotehniška fakulteta 0 0 0