20.500.12556/DKUM-64829 Optimizacija metode za ocenjevanje antioksidativne aktivnosti z uporabo barvila dcfh-da na celični liniji tlt Method optimization for tlt cell line based antioxidant activity assay using dcfh_da flurescent probe Na modelni makrofagni človeški celični liniji TLT smo optimizirali celično metodo z uporabo barvila DCFH-DA (2´, 7´-diklorofluorescin-diacetat) za določevanje pro- in antioksidativne aktivnosti modelnih substanc vitamina C in troloksa. Ugotavljali smo najprimernejše število celic na vodnjak, optimalen inkubacijski čas in koncentracijo testnih substanc ter najprimernejši vrstni red nanosa reagentov. Vrednost fluorescence v testu z DCFH-DA je bila linearno odvisna od števila celic na vodnjak, vendar le v odsotnosti oksidanta t-BuOOH. Pri eksperimentih se lahko uporablja širok interval koncentracije celic, saj so vse vrednosti izmerjene fluorescence še vedno bile v območju detekcije inštrumenta. Optimalen inkubacijski čas troloksa je od 1 do 2 uri, vitamina C pa 6 ur ali več. Visoke koncentracije obeh antioksidantov lahko vplivajo na nastalo fluorescenco, če sta prisotna v fazi merjenja. Optimalna vrstna reda nanosa reagentov sta bila testna substanca - DCFH-DA - t-BuOOH in t-BuOOH - testna substanca – DCFH-DA. We have optimized a TLT cell-based method using 2´,7´-dichlorofluorescin-diacetate (DCFH-DA) to assess the pro- and antioxidative activity of model substances vitamin C and trolox. We tried to determine the most appropriate number of cells per well, optimal incubation time and concentration of tested substances and the most appropriate order of reagent application. The fluorescence value in DCFH-DA assay was linearly dependent on the number of cells per well, but only in absence of the oxidant t-BuOOH. A wide range of cell concentrations can be used in this assay, that were within limits of instrument sensitivity. Optimal incubation time for trolox was 1 to 2 hours, while 6 or more hours are required for vitamin C. If those antioxidants are present while the measurement is conducted, their high concentrations can influence the resulted fluorescence. The most appropriate orders of reagent application were tested substance - DCFH-DA - t-BuOOH and t-BuOOH - tested substance - DCFH-DA. antioksidativna aktivnost DCFH-DA celice vitamin C troloks antioxidant activity DCFH-DA cells vitamin C trolox true true false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2016-12-20 14:36:28 2017-01-12 09:24:03 2022-07-06 03:06:46 2019-07-11 14:58:47 2016 0 Maribor 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 URN:SI:UM:DK:PIRNOT6A MAG_Oswald_Barbara_2016.pdf MAG_Oswald_Barbara_2016.pdf 1 1ABE58EBE77D941AC32493E61F898731 82b532809430abc8de239e3f547fa41ac5563b83bd976d16faa0b01810e8a970 e7b02b1d-7c0d-11eb-bb7a-00155d0001ca https://dk.um.si/Dokument.php?lang=slv&id=108819 Fakulteta za kmetijstvo in biosistemske vede 0 0 0