Quantifying Proliferating Lymphatic Endothelial Cells

LNs were digested as described above [39 (link), 40 (link)] or with Collagenase D (Worthington) and DNAse I (BD Biosciences) as described [2 (link), 41 ]. The cells from each mouse were plated into a 4-well chamber slide (Lab-tek, Nunc) in DME (Invitrogen) plus 10% fetal calf serum (Hyclone). After 24 h, non-adherent cells were removed and the remaining stromal cells were cultured in the presence of 30 μg/ml 10.1.1 Ab or control Hamster IgG for 5 days. Cells were stained with anti-Prox1 and anti-Ki67 Abs to identify proliferating LECs. Cells in at least 6 random 20x fields from each chamber were counted. Six mice were analyzed for each Ab treatment. Significance was determined using a Wilcoxon Ranked Sum test for paired samples using Prism (GraphPad Software).