Separation of hexose-transporting from nontransporting LLC-PK1 cells on density gradients

Over a period of 2-3 wk after plating, cultured LLC-PK1 (pig kidney) cells develop a high capacity for Na+-dependent accumulation of alpha-methyl-D-glucoside. To further the analysis of this developmental process, we have developed a method for separating transporting from nontransporting cells on the basis of density changes accompanying hexose accumulation and the corresponding uptake of water. Volume regulation was prevented by suspending the cells in a K+-free, Cl(-)-free Na-gluconate medium. Na+-dependent transport was maintained at nearly control levels by addition of low concentrations of (NH4)2SO4, since NH+4 stimulates Na+-K+-ATPase at the K+ site and allows for the extrusion of accumulated Na+; NH+4-stimulated hexose uptake is ouabain sensitive. With volume regulation blocked but with transport near normal, transporting cells exhibited a phlorizin-sensitive density shift in methylglucoside-containing medium and could be separated from nontransporting cells on Percoll gradients.