Fig. 3. Expression of vascular and lymphatic endothelial markers by BAE cells and LECs. (A) BAE cells and LECs were analyzed by FACS for the expression of VEGFR-3, VEGFR-2 and Prox-1. The gating was set using cells stained with isotype-matched human monoclonal antibodies (for VEGFR-2 and VEGFR-3) and a rabbit antibody to GFP (for Prox-1). LECs were strongly positive for both VEGFR-3 and VEGFR-2. About half of the BAE cells expressed significant surface VEGFR-3 as shown by staining with hF4-3C5, whereas the entire BAE cell population was positive for VEGFR-2. Staining of BAE cells for Prox-1 resulted in a minor shift relative to control antibody (RFI=5) whereas LECs were strongly positive for this marker (RFI=32). (B) cDNA obtained from BAE cells and LECs was analyzed by PCR for expression of lymphatic markers. Primers used in this analysis were located in regions of sequence identity in bovine and human sequences. LECs strongly express the lymphatic markers podoplanin (lane 4) and Prox-1 (lane 5). No expression of podoplanin (lane 1) or Prox-1 (lane 2) is detected in the cDNA from BAE cells. The control sequence G3PDH was amplified from both BAE cell and LEC cDNA. The lower panel shows the result of identical PCR reactions using cDNA prepared in the absence of reverse transcriptase. The predicted lengths of the amplified fragments are: podoplanin, 357 bp; Prox-1, 163 bp; and G3PDH, 261 bp. Together, the results of A and B demonstrate that the VEGFR-3-positive population of BAE cells is not lymphatic in origin.