LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian cells

Protocol tips

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	Add ethidium-calcein mixture, and incubate for 30 min in a 37 °C incubator

Protocol tips
Add ethidium-calcein mixture, and incubate for 30 min in a 37 °C incubator

Publication protocol

Cells for confocal studies were plated on chamber slides (5 × 104 cells/well) and infected with recombinant adenoviruses (100 PFUs/cell) for examination at 36 or 48 h. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature followed by incubation in methanol for 20 min on ice. After three washes, cells were permeabilized with 1% Triton X-100-PBS and further blocked with 5% goat serum. Immunofluorescence staining used the following primary antibodies: mouse monoclonal anti-FLAG (1:1000 dilution, Sigma-Aldrich), rabbit polyclonal anti-p65/RelA (1:200 dilution, Santa Cruz Biotechnology Inc.), and rabbit polyclonal anti-lamin B1 (1:200 dilution, Cambridge, Abcam). Secondary antibodies were either Alexa Fluor 488-labeled goat anti-mouse IgG (1:500 dilution, Invitrogen) or Alexa Fluor 546-labeled goat anti-rabbit IgG (1:500 dilution, Invitrogen) as appropriate. Cell nuclei were stained with DAPI using VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA). In some studies, cells were prestained with MitoTracker Red CMXRos (Invitrogen) for 30 min in culture medium before fixation. TUNEL staining used the DeadEnd fluorometric TUNEL assay (Promega). The pDsRed modified red fluorescent protein-p65/RelA expression plasmid was previously described (19). Cell viability assays used the LIVE/DEAD cell viability kit (Life Technologies). Fluorescence was analyzed using a Nikon C1si D-eclipse confocal microscope system and camera (Nikon Instruments, Melville, NY) with a Nikon plan Apo VC 60×/1.40 oil objective.

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