کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8269964 | 1534966 | 2014 | 15 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Response properties of the genetically encoded optical H2O2 sensor HyPer
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کلمات کلیدی
tert-butylhydroperoxideFCSYFPaCSFDAPITPLSMDTTGFPsHGIRFTCSPCtBHProGFPDEDTCCCD1,4-dithio-dl-threitol - 1،4-dithio-dl-threitol4′,6-diamidino-2-phenylindole dihydrochloride - 4 '، 6-دی آمیدین-2-فنیلینول دی هیدروکلرایدFLIM - FL منROS - ROSFluorescence resonance energy transfer - انتقال انرژی رزونانس FluorescenceFRET - انتقال انرژی رزونانسی فورسترInstrument response function - تابع پاسخ ابزارfluorescence lifetime imaging - تصویربرداری طولانی مدت فلورسنتOxidative stress - تنش اکسیداتیوSecond harmonic generation - تولید هارمونیک دومCharge-Coupled Device - دستگاه متصل شارژDiethyldithiocarbamic acid - دی اتیل دی اتیل کربام اسیدDIV - دیوFree radicals - رادیکال آزادdays in vitro - روز in vitroSOD - سدfetal calf serum - سرم گوساله جنینSuperoxide dismutase - سوکسوکس دیسموتازRedox signaling - سیگنالینگ Redoxendoplasmic reticulum - شبکه آندوپلاسمی time-correlated single-photon counting - شمارش تک فوتون همبستگی زمانیartificial cerebrospinal fluid - مایع مغزی نخاعی مصنوعیfluorescence lifetime imaging microscopy - میکروسکوپ تصویربرداری طول عمر فلورسنتTwo-photon microscopy - میکروسکوپ دو فوتونyellow fluorescent protein - پروتئین فلورسنت زردgreen fluorescent protein - پروتئین فلورسنت سبزReactive oxygen species - گونههای فعال اکسیژن
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
سالمندی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Reactive oxygen species mediate cellular signaling and neuropathologies. Hence, there is tremendous interest in monitoring (sub)cellular redox conditions. We evaluated the genetically engineered redox sensor HyPer in mouse hippocampal cell cultures. Two days after lipofection, neurons and glia showed sufficient expression levels, and H2O2 reversibly and dose-dependently increased the fluorescence ratio of cytosolic HyPer. Yet, repeated H2O2 treatment caused progressively declining responses, and with millimolar doses an apparent recovery started while H2O2 was still present. Although HyPer should be H2O2 specific, it seemingly responded also to other oxidants and altered cell-endogenous superoxide production. Control experiments with the SypHer pH sensor confirmed that the HyPer ratio responds to pH changes, decreasing with acidosis and increasing during alkalosis. Anoxia/reoxygenation evoked biphasic HyPer responses reporting apparent reduction/oxidation; replacing Clâ exerted only negligible effects. Mitochondria-targeted HyPer readily responded to H2O2-albeit less intensely than cytosolic HyPer. With ratiometric two-photon excitation, H2O2 increased the cytosolic HyPer ratio. Time-correlated fluorescence-lifetime imaging microscopy (FLIM) revealed a monoexponential decay of HyPer fluorescence, and H2O2 decreased fluorescence lifetimes. Dithiothreitol failed to further reduce HyPer or to induce reasonable FLIM and two-photon responses. By enabling dynamic recordings, HyPer is superior to synthetic redox-sensitive dyes. Its feasibility for two-photon excitation also enables studies in more complex preparations. Based on FLIM, quantitative analyses might be possible independent of switching excitation wavelengths. Yet, because of its pronounced pH sensitivity, adaptation to repeated oxidation, and insensitivity to reducing stimuli, HyPer responses have to be interpreted carefully. For reliable data, side-by-side pH monitoring with SypHer is essential.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Free Radical Biology and Medicine - Volume 76, November 2014, Pages 227-241
Journal: Free Radical Biology and Medicine - Volume 76, November 2014, Pages 227-241
نویسندگان
Jonathan Weller, Kathrin M. Kizina, Karolina Can, Guobin Bao, Michael Müller,