SEPARATION OF PROXIMAL TUBULE CELLS FROM SUSPENSIONS OF RAT-KIDNEY CELLS IN DENSITY GRADIENTS OF FICOLL IN TISSUE-CULTURE MEDIUM

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  • 1 January 1977
    • journal article
    • research article
    • Vol. 86  (3) , 591-602
Abstract
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 0.052 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.