Fig. 2
Both 55 °C and 85 °C HS cause instant and irreversible PM permeabilization. a Green fluorescence in the BY-2 cells loaded with FDA. Fluorescein leaks into extracellular space within 10 min after HS at either 55 °C or 85 °C. Arrows indicate shrunken protoplasts in 55 °C HS-treated cells. b Similar fraction of fluorescein leaks out of cells after 10 min of 55 °C, 85 °C or liquid nitrogen (N2) treatment. c BY-2 cells stained with SO and FM4-64 to assess whether disruption of the PM integrity after pulse HS is transient or irreversible. Staining was performed for no HS, before HS or after HS treatments according to protocols i, ii and iii, respectively (see “Sytox Orange and FM4-64 staining”). The cultures were imaged within 1 h following HS. d Frequency of the SO-positive cells in the cultures shown in c. DIC, differential interference contrast microscopy. IQR, interquartile range. Scale bars in a and c, 50 μm. b Representative data from one out of three independent experiments. d Data from three independent experiments, with ≥ 115 cells counted per treatment. b, d One-way ANOVA with Dunnet’s test; *p < 0.005