Live-cell imaging was carried out as described previously (12, 27, 63, 64). Briefly, indicated cells were grown on confocal dishes (Genetix Biotech Asia Pvt. Ltd.). All the plasmid DNA were transfected as above and examined by live-cell confocal microscopy, as indicated. Nuclei were stained with Hoechst 33342 (Sigma-Aldrich). Fluorophores were excited using either separately or in combination with ultraviolet (Diode 405), 488-nm (argon), 561-nm (DPSS 561), or 633-nm (HeNe 633) laser lines using a confocal laser scanning microscope (Leica TCS SP8) with 63×/1.4 numerical aperture oil objective equipped with a heated environmental chamber set to 37°C with an optimal CO2 facility. The percentage of cells displaying the indicated fluorescence was determined using Adobe Photoshop 7.0 from at least 20 to 25 cells expressing individual constructs.

Immunofluorescence staining and confocal microscopy were performed as described previously (12, 27, 28, 63, 64). Briefly, cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Primary antibodies against γH2AX were detected with anti-mouse IgG secondary antibodies labeled with Alexa Fluor 488 (Invitrogen). Cells were mounted in anti-fade solution with 4′,6-diamidino-2-phenylindole (Invitrogen) and examined using a laser scanning confocal microscope (Leica TCS SP8). Images were collected and processed using the Leica LAS X software and sized in Adobe Photoshop 7.0. The γH2AX intensity per nucleus was determined using Adobe Photoshop 7.0 by measuring the fluorescence intensities normalized to the number of cell count.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.