Figure 5.

Viability and proliferation of HHP-treated cells. (a) Representative histograms of Cytocalcein 450 fluorescence (viability stained) among groups, with cells exposed to 50 MPa for 36 and 48 h showing negative expression, similar to STS treatment, while those pressurized for 24 h show bimodal distribution. (b) The comparison diagram shows a significant reduction in the Cytocalcein MFI between the 0 MPa group and the group treated with 50 MPa for 24, 36 or 48 h (n = 3). (c) A flow cytometry analysis showing two distinct cell populations depending on the cell size (FSC) and granularity (SSC). The oval area in the control group indicates normal viable cells, which are present in the positive control (STS) group at only 0.52%. That number in the 0 MPa group is around 82% while viable cells account for around 41.3% after being pressurized at 50 MPa for 24 h and 9.73% and 6.32% after 36 and 48 h, respectively. Data are representative of at least three independent experiments. (d) Absorbance at 450 nm in the WST-8 assay shows the proliferation of control, untreated, HHP-treated and STS-treated cells after 7 days of seeding culture. Data are representative of at least two independent experiments, n = 7 for each time point. (e) Inverted light microscopy images of untreated, HHP-treated and STS-treated cells seeded onto a 24-well plate after 7 days of culture. The white arrow in the 50 MPa_24h group indicates viable cells. Magnification 10×, scale bar 100 μm. Data are representative of at least three independent experiments.