Pi double staining kit

Cell viability was measured using a calcein-acetoxymethyl (AM)/propidium iodide (PI) double staining kit (Dojindo, Kumamoto, Japan). In brief, cells were fixed with 70% ethanol for 15 min. They were then stained with calcein-AM solution (10 μmol/l) for 15 min. They were then washed with phosphate-buffered saline (PBS) buffer and stained with PI (10 μmol/l) for 15 min. The optical density at 490 nm and 535 nm were determined to determine healthy cells and dead cells, respectively.

To
investigate the effects of [Fe(CN)₆]^4– on HUVEC monolayers, VE-cadherin in the HUVEC monolayers was stained.
First, HUVECs were cultured in a 24-well culture plate (Corning) for
7 days to prepare HUVEC monolayers. The monolayers were washed with
PBS three times and then immersed in PBS containing 4% paraformaldehyde
(Fujifilm Wako Pure Chemical Co., Japan) for 15 min. Next, after washing
with PBS three times, the monolayers were immersed in 0.1% Triton
X-100 (Sigma-Aldrich Japan, Japan) for 15 min. After removing Triton
X-100, the monolayers were immersed in PBS containing 1% bovine serum
albumin (Sigma-Aldrich Japan) and rabbit anti VE-cadherin antibody
(1:500 dilution, Cell Signaling Technology, USA). After incubating
overnight at 4 °C, the solution was replaced with PBS containing
a secondary antibody, Alexa Fluor 647 conjugated goat anti-rabbit
IgG (1:1000 dilution, Thermo Fisher Scientific, USA), and 45 μM
propidium iodide (PI, double staining kit, Dojindo, Japan). The monolayers
were incubated overnight at 4 °C and then observed under a fluorescence
microscope (Leica DMi8, Leica, Germany).