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American Journal of Pathology, Vol 86, 591-602, Copyright © 1977 by American Society for Investigative Pathology

REGULAR ARTICLES

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

JI Kreisberg, AM Pitts and TG Pretlow

Rat kidneys were disaggregated with 0.25% trypsin. Cell were separated by velocity sedimentation in a previously described isokinetic gradient, by isopycnic sedimentation, and by velocity sedimentation followed by isopycnic sedimetation. 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 border suggested that HDAP might be a suitable marker for rat proximal tubule cells. These cell 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 typan blue. Following isopycnin centrifugation, the purest fractions contained 87.3+/-1.5% nucleated cells with HDAP, 9.6+/- 2.5% nucleated cells iwithout 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 cell with HDAP, 6.0+/- 2.3% nucleated cells with HDAP, and 1.0+/-0.9% red blood cells. These proximal tubule cells sedimented a density of 1.041 g/ml. More than 98% of these cells excluded trypan blue.




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Copyright © 1977 by the American Society for Investigative Pathology.