Generation of primary cultures of murine podocytes or parietal cells.

<p>A. First capsulated or decapsulated glomeruli were purified using magnetic beads (arrow with tails, >90% purity), to prepare primary mixed podocyte or parietal cell cultures respectively. For direct comparison, a capsulated glomerulus (arrows) is shown next to a decapsulated glomerulus (arrowheads). B, C. After 7–14 days of culture in EGM-MV media, primary outgrowths were obtained and stained for the genetic marker (X-gal). D, E. Next, suspensions of primary cells were treated with a luminescent substrate for beta-galactosidase (“labeled”, D′, E′) or not (controls, D, E) and were FACS-sorted using the indicated gate (“target cells”). In the example shown, 35,2% of the cells were defined as podocytes (D″) and 6.6% of the cells were defined as parietal cells (E″). F, G. The purity of enriched primary podocytes (F, F′) and parietal cells (G, G′) was evaluated after six passages in culture. Staining for the genetic marker beta-gal (X-gal) revealed that virtually all of the cells were either derived from podocytes or parietal cells (merged images: phase contrast+X-gal stainings). H, I. Similarly, in a FACscan analysis, virtually all cells were positive for the genetic tag beta-galactosidase (as revealed by metabolic labeling with FDG). In this representative analysis, 97.1% and 99.4% of the primary podocytes or PECs were defined as beta-gal positive, respectively.