Quantifying Apoptosis in Co-cultured Cells using TUNEL Assay

For the fluorescein terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, we followed the manufacturer’s protocol [11 (link),22 (link)]. All samples were thoroughly rinsed with PBS for 5 min three time before the assay. After antigen retrieval and blocking, slides were rinsed twice with PBS and excess fluid drained. The TUNEL reaction mixture was added and incubated with labeling solution for 60 min at 37 °C, protected from light. Negative controls were incubated with labeling solution only. Nuclei were stained with DAPI. Fluorescent images were reviewed using an Olympus BX51-microscope prior to further analysis using a Nikon A1R MP ECLIPSE Ti confocal microscope, with NIS-Elements imaging software version 4.50.00 (Nikon, Tokyo, Japan). A published protocol was used to do quantitative analysis of the extent of TUNEL signal-positive cells in the co-cultures of Vero cells and HUVECs. Briefly, TUNEL and DAPI signals were captured with an Olympus BX51 image system using a final 10× optical zoom. The extent of TUNEL signal-positive cells is normalized to total nuclear content (DAPI staining) in each filed using NIH ImageJ [66 (link)]. Twenty-six microscopic fields were examined for each sample. Data are representative of at least three experiments.