Click it tunel alexa fluor 594 imaging assay

1.5 × 10³ GFP-expressing tumor cells were added onto the endothelial monolayer and cultured overnight. Cells were then fixed for 15 min in 4% PFA at room temperature. Apoptotic cells were detected using the Click-iT TUNEL Alexa Fluor 594 Imaging Assay (C10246; Invitrogen) following the manufacturer’s instructions.

Purified islets were washed twice with KRB 5.6 mmol/L glucose and 3% BSA, fixed with 4% paraformaldehyde for 4 min in 0.1 mol/L PBS, washed with PBS and permeabilized with 0.02% Triton X-100 overnight. The primary antibody was an anti-insulin mouse monoclonal antibody (1:200 dilution; Sigma-Aldrich, St. Louis, MO, USA). The primary antibody localization was detected using anti-mouse TRITC (1:300 dilution; Sigma-Aldrich, St. Louis, MO, USA). Apoptosis was measured using a TUNEL staining in situ cell death kit (click-it Tunel Alexa Fluor 594 Imaging Assay; Invitro gen, Madrid, Spain). Proper controls for the secondary antibodies revealed no nonspecific staining. Nuclei were counterstained with 300 nM DAPI (Sigma Aldrich, Madrid, Spain). Confocal fluorescence images were captured with a Leica TCS SP5 confocal microscope (Leica, Wetzlar, Germany) at 2-µm intervals in the z-dimension. Images were analyzed using the open source image processing package FIJI. The β-cell number per islet was quantified by counting the number of cell nuclei within the insulin immunoreactive area. The apoptosis results are expressed as the number of TUNEL-positive β-cells over the total number of cells.

For immunofluorescence and TUNEL assay, 3 × 10⁴ cells were plated in chambers slides (Millipores, USA), fixed in 4% paraformaldehyde for 10 min, and then permeabilized in 0.5% Triton X-100-phosphate buffered saline (PBS) for 15 min. Cell death was measured by using Click-it TUNEL Alexa Fluor® 594 Imaging Assay (Invitrogen, UK) according to manufacturer’s instructions followed by blocking with BSA 3% in PBS for 1 h. Incubation with E-cadherin primary antibody for 1 h was followed by incubation in Alexa-Fluor 488-conjugated secondary antibody solution for 1 h. To visualize nuclei, it was used 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, LifeTech, UK).Finally, the mounting media used was ProLong Gold antifade reagent (LifeTech, USA). Epifluorescence images were taken in Olympus microscope.