Immediately after 24 h, nuclear morphology of 300 cells per sample was observed to investigate the presence of micronuclei and double nuclei. Fluorescence microscopy of residing cells ROS formation and results on mitochondria were ana lysed in living cells applying DCFH DA, MitoTracker and MitoSOX dyes. ROS and mitochondria co localization was investigated soon after two h of PM therapy. Cells grown on cover slips were initially incubated at 37 C with five uM of DCFH DA in PBS for 20 min, then exposed to PM and eventually stained with MitoTracker for thirty min and counter stained with natural product library DAPI. Slides were observed underneath a fluores cence microscope, digital pictures had been taken by using a last magnification of 630? and co localization signal was quantified with Axiovision Rel four.
8 co localization devoted computer software, Images of mitochondria stained with MitoTracker were also taken following 24 h of remedy with PM, to investigate attainable secondary results. Ultimately, the formation of mitochon drial superoxide was selleck examined by staining the cells with MitoSOX. Briefly, after 2 and 24 h of PM treatment, cells grown on cover slips have been loaded with two uM Mito SOX functioning alternative for 15 min at 37 C, inside the dark. Then, cells had been washed in HBSS Ca Mg and fixed with 3% paraformaldehyde for 15 min. Digital photographs have been taken by a fluorescence microscope using a ultimate magnifi cation of 630?, Western blotting The expression levels of p53 and Chk2, and of their ac tive phosphorylated forms pp53 and pChk2, have been ana lyzed by Western blotting to assess their involvement in cell cycle regulation. Just after 3 and ten h of exposure to winter PM2.
five, cells were collected, washed in PBS and stored overnight at 80 C. Cells had been lysed in RIPA buf fer, sonicated 3 occasions for thirty sec on ice and ultimately homogenised applying a syringe needle. Cell lysates had been then separated by SDS Page on 10% gels and transferred to nitrocellulose membranes. Blots have been incubated with ideal anti bodies overnight at 4 C. Just after washes, the membranes were incubated with HRP linked secondary antibodies and subsequently incubated with Chemilumin escent Peroxidase Substrate for detec tion. Digital photos have been taken by a luminescence reader and densitometry examination was carried out with dedicated application, Information were normalized to the actin information and expressed as fold maximize over manage. DNA damage Single cell gel electrophoresis Immediately after one h publicity to antioxidants and inhibitors and three h publicity to PM, media had been eliminated and cells trypsinized and resuspended at one million cells ml in PBS. Samples were analysed for DNA strand breaks and alkali labile internet sites employing the comet assay.