2.6. Cell Membrane Permeability Assay

The effect of cranberry PACs on plasma membrane integrity of macrophages was assessed using the intracellular dye calcein acetoxymethyl ester (calcein-AM) (Sigma-Aldrich Canada). Briefly, macrophages were differentiated in a black walled, black bottomed, 96-well microplate (Greiner Bio-One North America Inc., Monroe, NC, USA), and were treated with LtxA (1 µg/mL) in the absence or presence of two-fold serial dilutions of cranberry PACs (125 to 7.9 µg/mL in phosphate-buffered saline (PBS; pH 7.2)) in the presence of 5 µL of 1 mM calcein-AM for 3 h at 37 °C in a 5% CO₂ atmosphere. Calcein-loaded macrophages were observed using an inverted Olympus FSX100 fluorescent microscope (Olympus Canada Inc., Richmond Hill, ON, Canada).