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

J. I. Kreisberg, A. M. Pitss, T. G. Pretlow

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

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 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.

Original languageEnglish (US)
Pages (from-to)591-602
Number of pages12
JournalAmerican Journal of Pathology
Volume86
Issue number3
StatePublished - 1977
Externally publishedYes

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

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