Separation of proximal tubule cells from suspensions of rat kidney cells in density gradients of ficoll in tissue culture medium

Rat kidneys were disaggregated with 0.25% trypsin. Cells were separated by velocity sedimentation in a previously described isokinetic gradient, by isopycnic sedimentation, and by velocity sedimentation followed by isopycnic sedimentation. In some fractions from the isokinetic gradient, 71.8 ± 2.4% of the nucleated cells contained histochemically demonstrable alkaline phosphatase (HDAP); in semithin sections, 62.7 ± 2.3% of these cells had brush borders. The correspondence between fractions enriched for cells with HDAP and fractions enriched for brush borders suggested that HDAP might be a suitable marker for rat proximal tubule cells. These cells constituted 46.5 ± 2.6% of the nucleated cells in the starting sample suspension of kidney cells, and 81.9 ± 2.7% of nucleated cells in the purified fractions from the gradients. More than 98% of nucleated cells in these fractions excluded trypan blue. Following isopycnic centrifugation, the purest fractions contained 87.3 ± 1.5% nucleated cells with HDAP, 9.6 ± 2.5% nucleated cells without HDAP, and 3.1 ± 2.5% red blood cells. These proximal tubule cells had densities of 1.036 to 1.052 g/ml. With rate zonal separation followed by isopycnic separation, the purest gradient fraction contained 93.0 ± 1.9% nucleated cells with HDAP, 6.0 ± 2.3% nucleated cells without HDAP, and 1.0 ± 0.9% red blood cells. These proximal tubule cells sedimented to a density of 1.041 g/ml. More than 98% of these cells excluded trypan blue.