Simultaneous 1H PFG-NMR and confocal microscopy of monolayer cell cultures: effects of apoptosis and necrosis on water diffusion and compartmentalization.

We induced apoptosis and necrosis in monolayer cultures of Chinese hamster ovary cells using okadaic acid and hydrogen peroxide (H2O2), respectively, and examined the effect on water diffusion and compartmentalization using pulsed-field-gradient (PFG) 1H-NMR and simultaneous confocal microscopy. In PFG experiments characterized by a fixed diffusion time (<4.7 ms) and variable b-values (0-27000 s/mm2), 1H-NMR data collected with untreated cells exhibited multiexponential behavior. Analysis with a slow-exchange model revealed two distinct cellular water compartments with different apparent diffusion coefficients (ADCs; 0.56, 0.06 x 10(-3) mm2/s) and volume fractions (0.96 and 0.04). During the first 12 hr of necrosis or apoptosis, the amount of water in the smallest compartment increased twofold before significant changes in cell density or plasma membrane integrity occurred. Over the same period, water content in the largest compartment decreased by a factor of >2 in apoptotic cells, in accordance with observed cell shrinkage, and changed little in necrotic counterparts, where only slight swelling was evident. These results indicate that PFG 1H-NMR serves as a sensitive indicator of early cell death in monolayer cultures, and can be used to distinguish apoptosis from necrosis. Measurements of restricted diffusion and water exchange are presented to elucidate the compartment origins and justify the model assumptions.