Fluorescence microscopy of living cells ROS formation and results on mitochondr

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Fluorescence microscopy of living cells ROS formation and results on mitochondr

Post  jy9202 on Thu Mar 03, 2016 4:17 am

Fluorescence microscopy of living cells ROS formation and results on mitochondria were ana lysed in living cells making use of DCFH DA, MitoTracker and MitoSOX [You must be registered and logged in to see this link.] dyes. ROS and mitochondria co localization was investigated following two h of PM remedy. Cells grown on cover slips had been initial incubated at 37 C with 5 uM of DCFH DA in PBS for twenty min, then exposed to PM and finally stained with MitoTracker for 30 min and counter stained with DAPI. Slides have been observed under a fluores cence microscope, digital pictures had been taken which has a ultimate magnification of 630and co localization signal was quantified with Axiovision Rel four. 8 co localization committed application. Photographs of mitochondria stained with MitoTracker had been also taken following 24 h of treatment method with PM, to investigate possible secondary results.

Ultimately, the formation of mitochon drial superoxide was examined by staining the cells with MitoSOX. Briefly, following two and 24 h of PM therapy, cells grown on cover slips have been loaded with two uM Mito SOX working answer for 15 min at 37 C, from the dark. Then, cells had been washed in HBSSCaMg and fixed [You must be registered and logged in to see this link.] with 3% paraformaldehyde for 15 min. Digital photos were taken by a fluorescence microscope using a final magnifi cation of 630. Western blotting The expression levels of p53 and Chk2, and of their ac tive phosphorylated forms pp53 and pChk2, had been ana lyzed by Western blotting to assess their involvement in cell cycle regulation. After 3 and 10 h of publicity to winter PM2. 5, cells were collected, washed in PBS and stored overnight at −80 C.

Cells were lysed in RIPA buf fer, sonicated 3 occasions for 30 sec on ice and ultimately homogenised making use of a syringe needle. Cell lysates were then separated by SDS Page on 10% gels and transferred to nitrocellulose membranes. Blots have been incubated with proper anti bodies overnight at four C. Soon after washes, the membranes have been incubated with HRP [You must be registered and logged in to see this link.] linked secondary antibodies and subsequently incubated with Chemilumin escent Peroxidase Substrate for detec tion. Digital photographs have been taken by a luminescence reader and densitometry evaluation was performed with focused software program. Information had been normalized towards the actin material and expressed as fold maximize more than manage.

DNA injury Single cell gel electrophoresis Just after 1 h publicity to antioxidants and inhibitors and three h exposure to PM, media had been removed and cells trypsinized and resuspended at one million cellsml in PBS. Samples had been analysed for DNA strand breaks and alkali labile sites applying the comet assay. Cells dissolved in 0. 68% LMP agarose in PBS with ten mM EDTA, pH seven. four, had been moulded onto GelBond movies connected to plastic frames to facilitate subsequent techniques. Movies underwent lysis overnight at 4 C, and then have been transferred to cold electrophoresis resolution for 40 min at 4 C for DNA unwinding. Soon after electrophoresis and neutralisation, films had been fixed in ethanol and dried. Rehydrated samples have been stained with SybrGold and scored with Perceptives Comet IV computer software. The degree of DNA injury was expressed as tail intensity, i. e. percent fluorescence inside the comet tail, relative to the comet complete fluorescence. 32P postlabelling DNA adducts were measured from the thin layer chromatog raphy 32P postlabelling strategy utilizing the nuclease P1 digestion enrichment model from the assay.


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