Fluorescein isothiocyanate-labeled annexin V was obtained from R&D Systems Inc. (Minneapolis, MN, USA) and PromoKine (Heidelberg, Germany). Annexin V binds to phosphatidylserine; therefore, we used annexin V to detect the lipid flip-flop induced by 30 minutes nisin treatment.
The cells were incubated with annexin V for 15 minutes in the dark according to a previous study and the manufacturer’s instructions [30 (link), 44 (link)]. Cells were washed several times to remove unbound annexin V. Images of paraformaldehyde-fixed cells and non-fixed cells were acquired and automatically tiled with a BZ-X700 fluorescence microscope (Keyence, Osaka, Japan) using a 20× or 40× objective with numerical apertures of 0.45, and 0.95, respectively. The ratio of annexin V-positive cells was analyzed using the fixed-cell images. Images were analyzed using the hybrid cell count function of the BZ-X analyzer (Keyence).
The cells were incubated with annexin V for 15 minutes in the dark according to a previous study and the manufacturer’s instructions [30 (link), 44 (link)]. Cells were washed several times to remove unbound annexin V. Images of paraformaldehyde-fixed cells and non-fixed cells were acquired and automatically tiled with a BZ-X700 fluorescence microscope (Keyence, Osaka, Japan) using a 20× or 40× objective with numerical apertures of 0.45, and 0.95, respectively. The ratio of annexin V-positive cells was analyzed using the fixed-cell images. Images were analyzed using the hybrid cell count function of the BZ-X analyzer (Keyence).
