1 Biofyzikálni ústav AV CR - Mozilla FirefoK JflJxJ Soubor Úpravy Zobrazení Historie Záložky Nástroje Nápověda C ŕ4 líLi \ÍSP http:jiwww.ibp.cz/cs/oddeleni/patofyziologie-volnych-radikalu/zamestnanci/ Ú - |ťl'| Google Ji_ Nejnavštěvovanější ^ Centrála Firefoxu Přehled zpráv Bentham Science Publ... tBP Biofyzikálni ústav AV ČR Zaměstnanci oddělení Patofyziologie volných radikálů Vedoucí oddelení Lojek Antonín, doc. RNDr.. CSc. faloiek@jbp.ozl Vedoucí vědečtí pracovníci Číž Milan. RNDr.. Fh.D. f mi lancig@jbp.cz 1 Kubala Lukáš. Mgr.. Fh.D. f kubalal@jbp.csl Vědečtí pracovníci Feichalová Kateřina. Mgr.. Fh.D. fpeichalo vak@jbp.cz 1 Pekařova Michaela. Mgr.. Fh.D. f pe karová rn@j bp .cz 1 Yítecek Jan. Mgr.. Fh.D. f i an. vitece k@j bp .cz 1 Doktorandi Ambrožová Gabriela. Mgr. fa mbrozo va@i bp .cz 1 Eino Lucia. Mgr. fbino@jbp.czl Crhák Tomáš. Mgr. fcrhak@jbp.cz! Kolářová Hana. Mgr. f kol aro va@j bp .cz 1 Šafránková Barbora. Mgr. f saf ran ko va@j bp .cz 1 Vašíček Ondřej. Mgr. f ondre i .vaši ce k@j bp .cz 1 Studenti Gajdová Silvie. Bc. f ga i do va@j bp .cz 1 Marti š ková Hana. Bc. f marti s kova.h@ibp.cz 1 Navrátilová Jana. Bc. f na vráti I o va@i bp .cz 1 Rajecký Michal. Bc. fraieckv@ibp.czl Techničtí pracovníci Vystrčil o vá Lenka f vystrčí I o va@j bp .cz 1 1 Biofyzikálni ústav AV CR - Mozilla FirefoK Soubor Úpravy Zobrazení Historie Záložky Nástroje Nápověda T C i_Ej j IBP 1 http://www.ibp.cz/cs/oddeleni/patofyziologie-volnych-radikalu/metody/ ft ' |?'|-| Google Nejnavštěvovanější ^ Centrála Firefoxu [S] Přehled zpráv Bentham Science Publ... \SP Biofyzikálni ústav AV ČR vyuKd Mezinárodní projekty Národní projekty transplantation (heart, liver, kidney) or regular hemodialysis treatment. ethods: Parameter Method cell numbers coulter-counter, microscopy cell viability spectrophotometrv. flow cvtometrv. microscopy cell morphology microscopy cell cycle flow cvtometrv metabolic activity as a measure of the number of viable cells spectrophotometrv. luminometrv oxidative burst of neutrophils luminescence, flow cvtometrv MPO activity luminescence, spectrophotometrv NO synthesis luminescence, flow cvtometrv. spectrophotometrv total radical-trapping antioxidant parameter luminescence individual antioxidants spectrophotometrv lipid peroxidation spectrophotometrv surface molecule expression flow cvtometrv activity of specific enzymes luminometrv. spectrophotometrv Chemiluminescence methods Laboratory is equipped with cuvette (BioOrbit) and microtitre plate flmmunotech^ luminometers. Luminol- or lucigenin-enhanced chemiluminescence is used for the measurement of ROS Hotovo ifc Start | J ^ ® O W I glAbstractEng.doc- ... | g, Inbox - Microsoft 0... | g špeciálni metody ■ ppt | Biofyzikálni ústav ... J g |gEElQ<&0 gj© Í&^O 14:43 Luminometry, fluorimetry and electrochemistry in the analysis of phagocyte-derived reactive oxygen and nitrogen species and antioxidative capacity of biological samples Phagocyte-derived reactive oxygen and nitrogen species Assays used to measure ROS production • NBT test • Cytochrom c reduction assay • Oxygen uptake (Clark type electrodes) • Fluorescence • Chemiluminescence Luminometric methods enable: 1. continual analysis of the oxidative burst 2. to differentiate between the intra- and extracellularly generated reactive oxygen species. Orion II Microplate Luminometer Chemiluminescent Indicators • Lucigenin • Luminol • Isoluminol • Pholasin LUMINOL Luminol-enhanced CL reflects primarily myeloperoxidase activity Luminol-enhanced CL reflects reflects also production of other oxidants in physiological pH: .> Peroxynitrite > Hydrogen peroxide together with hypochlorite > Hydrogen peroxide together with heme iron (MPO, HRP) At alkaline pH, luminol is easily oxidised by week oxidants (hydrogen peroxide alone) ISOLUMINOL Differs from luminol only with respect to the position of the amino group in the phthalate ring of the molecule. It does not produce luminescence as efficiently as luminol. As published by several authors (e.g. Lundquist and Dahlgreen, 1996), isoluminol does not cross the plasma membrane. LUCIGENIN Lucigenin-dependent CL is myeloperoxidase independent and measures NADPH oxidase associated oxygenation, essentially superoxide. Criticism of superoxide-lucigenin method: in higher concentrations lucigenin itself can generate superoxide and confound the results. PHOLASIN Photoprotein of the bioluminescent mollusc (Pholas dactylus) Emits light in the presence of free radicals and oxidants. Pholasin is a 34 kDa protein that is impermeable to cells (Arnhold et al., 2002) Peak of CL (RLU) Final C (stock 10 mM in water) 1mM 0,1mM 0,01mM 1microM Luminol 163,33 116,67 26,17 27,67 izoluminol 111,33 43,50 13,33 12,17 lucigenin 30,17 30,67 20,33 12,50 Final C (stock 10 mM in DMSO) 1mM 0,1mM 0,01mM 1microM Luminol 44,17 185,83 39,17 14,17 izoluminol 83,67 49,17 15,33 11,17 lucigenin 27,00 35,17 19,17 12,00 Final C (stock 10 mM in borate buffer) 1mM 0,1mM 0,01mM 1microM Luminol 415,83 193,33 43,50 14,00 izoluminol 271,00 48,17 14,33 11,17 lucigenin 51,83 24,17 14,17 12,33 250 -200 -150 -100 -50 C 0 0 40 30 20 10 30 TIME (min) 50 40 30 20 10 0 D 30 TIME (min) 15 12 9 6 3 0 Lojek A., Kubala L., ČížováH., Číž M. (2002): A comparison of neutrophil chemiluminescence in cuvettes and microtittre plates. Luminescence, 17, 1-4. 0 Drábiková K., Nosál' R., Jančinová V., Číž M., Lojek A (2002).: Reactive oxygen metabolites production is inhibited by histamine and H1-antagonist dithiaden in human PMN-leukocyte. Free Rad. Res. 36(9), 975-980 Nosal R., Jancinova V., Ciz M., Drabikova K., Lojek A., Fabryova V.(2005): Inhibition of chemiluminescence by carvedilol in the cell-free system, whole human blood and blood cells. Scand J Clin Lab Invest 65, 55-64 Phagocyte-derived nitric oxide production Assays used to measure NO production • cell-permeable fluorescent indicators (4,5-diaminofluorescein diacetate (DAF-2 DA) • total nitrate/nitrite concentration • NO donor compounds, NO scavengers • NOS activity in cell homogenates by measurin g the enzymatic conversion of arginine to citrulline during NO formation • NOS inhibitors • antibodies to NOS isoforms by immunocytochemistry or by immunoblotting • electrochemical method for direct measurement of NO concentration Electrochemical method Duo* 18 Now records data directly from NOMKit EVOMX, IS02, pH electrodes and Ian Selective Electrodesi Micromanipulator M3301 ISO-NO Nilik Oxide Meter ■iLil'k- nút Included} Hrbac J, Gregor C, Machova M, Kralova J, Bystron T, Ciz M, Lojek A. (2007) Nitric oxide sensor based on carbon fiber covered with nickel porphyrin layer deposited using optimized electropolymerization procedure. Bioelectrochemistry. Sep 27; [Epub ahead of print] Fig. 2. Catbon fiber sensor's performance at nanomolar NO concentrations (CPA at K30 mV (vs. Ag/AgCL}}. Eight additions of NO into aeraled PBS, each resulting in 4 nM NO concenLralion, followed by response for 20 nM NO are shown. After pretreaiment the electrode was coated with poly-NiTMIIPP, electropolymeruzed foom 0.4 mM NiTMIIPP by 100 cycles from 0 to 1200 raV (vs. Ag/AgClK scan rale 100 mV/s). 2.0' 1.5 ^ 1.0« 0.5 0 5 RAW 264.7 without stimulation RAW 264.7 stimulated by LPS I 10 Time / h i 15 20 Time course of NO production by RAW 264.7 cell culture after stimulation (priming) by LPS 25 20 15 10 5 0- Time course of nitrite accumulation in the supernatant collected from RAW 264.7 cell culture 0 r 10 i 15 20 Time / h Fluorimetric and Luminometric determination of antioxidative activity Luminometric determination of antioxidative activity All individual antioxidants can be analysed at the same time using the measurement of otal Radical-trapping Antioxidative Potential TRAP - basic principle • 2,2'-azobis(2-amidinopropane) dihydrochloride • 2,2'-azobis(2-methylpropionamide) dihydrochloride TRAP - basic priciple RN == NR N2 + 2R* — 2RO2* peroxyl radical R2 + following reactions Orion II Microplate Luminometer 0 1020 3040 50 Čas [min] Infinite M200 Fluorimetrical assay [Prior and Cao, 1999]. OXYGEN RADICAL ABSORBANCE CAPACITY • Antioxidant scavenging activity against peroxyl radical induced by 2,2-azobis (2-amidinopropane)hydrochloride (ABAP) at 37°C • Fluorescein (FL) - fluorescent probe • The lost of fluorescence of FL is an indication of the extent of damage from its reaction with the peroxyl radical • The antioxidative effects of a drugs are measured by assesing the area under the fluorescence decay curve (AUC) Example: ORAC of antihistamine drug Astemizol Time [min] ORAC TRAP |jmol TE/g FW | mol TE/g FW 1 celery - leaves 113,5 ± 6,1 celery - leaves 68,14 ± 4,79 2 parsley 108,6 ± 13,1 parsley 67,15 ± 8,92 3 lovage 57,3 ± 5,0 lovage 28,41 ± 1,30 4 chilli pepper 36,1± 6,5 goathorn pepper 24,8 ± 1,18 5 goathorn pepper 30,6 ±1,4 chilli pepper 23,72 ± 3,06 6 radish 23,6 ± 1,7 capsicum 20,91 ± 1,15 7 capsicum 19,9 ± 1,4 red beet 17,85 ± 0,25 8 eggplant 16,2 ± 2,0 green bean 16,88 ± 0,45 9 broccoli 16,1± 1,2 dill 13,66 ± 1,50 10 celery - root 15,3 ±1,2 eggplant 13,40 ± 1,47 11 green onion 14,7± 1,5 radish 11,39 ± 2,24 12 gumbo 14,6 ± 0,8 gumbo 7,42 ± 0,77 13 green bean 14,5 ± 1,2 red pepper 5,93 ± 0,14 14 red beet 12,6 ± 1,6 tomato 4,35 ± 0,47 15 dill 10,5 ±1,1 potato 3,78 ± 0,10 16 potato 10,3 ±1,3 green onion 3,48 ± 0,52 17 red pepper 9,3 ± 0,9 broccoli 3,39 ± 0,12 18 green pepper 5,6 ± 0,3 green pepper 2,81 ± 1,88 19 tomato 5,4 ± 0,3 celery - root 1,00 ± 0,43 20 carrot 4,8 ± 1,1 carrot 0,00 21 vegetable marrow 2,9 ± 0,3 vegetable marrow 0,00 22 cucumber 1,2 ± 0,2 cucumber 0,00 ORAC [ |jTio! TEIg FW] Institute of Biophysics of the AS CR Department of Free Radical Pathophysiology