Elipse ti confocal microscope

Cells were either plated on glass bottom petriplates or were transferred to glass slides by cytospin after the appropriate specified treatment. The cells were fixed in 3.7% paraformaldehyde for 10 min followed by permeabilization for 5 min using 0.2% Triton X-100 in PBS. The cell were then washed twice and blocked with 10% goat serum and 1% BSA in PBS for 30 min at 37 °C followed by staining with the primary antibody (1:100 dilution, 1% BSA in PBS) for 2 h at 37 °C. After the incubation, the cells were washed three times (5 min) with PBS and incubated with secondary-TRITC labelled antibody for 1 h at 37 °C, washed three times with PBS, and mounted with DAPI (40,6-Diamidino-2-phenylindole dihydrochloride) for nuclear staining. Cells were visualized under a Nikon Elipse Ti confocal microscope and captured using NIS-elements imaging software. The experiments were repeated three to four times and the figures are representative maximum image projections with the same laser power settings.

To test apoptosis; apoptosis/necrosis detection kit from Abcam (Cambridge, MA) was used. The kit uses apopxin green indicator dye which binds to phosphatidylserine which is flipped outside during apoptosis. 7-AAD dye in the kit is able to detect late apoptotic and necrotic cells as it is a membrane impermeable dye and stains cells with loss of plasma integrity. Cells were trypsinized 72 h after the siRNA treatment and stained with apopxin and 7-AAD for 30 min at RT in assay buffer as described in the manufacturers protocol. The cells were analyzed using C6 Accuri flow cytometer. The apopxin dye is generally read in FL1 channel (Ex/Em = 490/525) whereas 7-AAD is read in the FL3 channel (Ex/Em = 550/650). Alternatively cells were grown on glass plates and after staining with apopxin and 7-AAD were analyzed using the Nikon Elipse Ti confocal microscope.