Fig. 4.

Characterization of the mechanism of cell death

THP-1 cells were seeded into 24-well plates at 10⁶ cells/mL in serum free RPMI media and were cultured for 4 h (A–D) or 24 h (E–H) at 37 °C in the presence of 50–200 mM NaCl (○), Arg·Glu (●), Arg·HCl (■) or NaGlu (∆). Control cells were cultured with medium alone. To characterize the extent and pattern of induced cytotoxicity, cells were stained with Annexin V-FITC (FL-1 channel) and PI (FL-2 channel) and 10,000 cells were analyzed using a FACSCalibur flow cytometer. The results are displayed as the percentages of cells that are Annexin V-ve/PI-ve (viable) (A, E), Annexin V + ve/PI-ve (early apoptotic) (B, F), Annexin V + ve/PI + ve (late apoptotic) (C, G), Annexin V-ve/PI + ve (necrotic) (D, H). In each case, data are shown as % total cells in each category (mean and SE for n = 3 experiments, where for clarity SE > 2% only are shown). The statistical significance of differences between cells cultured in medium alone and cells treated with various concentrations of salts was assessed by one way ANOVA (p < 0.05; * = NaCl, # = Arg·Glu, ¥ = Arg·HCl, δ = NaGlu).