Purification and GeneChip analysis of fresh BECs, LECs, and other major skin cell types. (A) Identification and isolation of fresh BECs and LECs by triple immunolabeling and FACS. Microvascular ECs in freshly prepared dermal cell suspensions were identified by their homogeneous VE-cadherin expression (y-axis, left). The sort gate was set to actively exclude VE-cadherin⁻ non-ECs and VE-cadherin⁺ dead ECs that are characterized by their low forward scatter (FSC) signals (x-axis). Within the vital EC population, nonoverlapping subpopulations of BECs (red dots) and LECs (green dots) were resolved by anti–podoplanin-FITC (x-axis) anti–CD34-PE immunolabeling (y-axis, right). BECs and LECs were purified to at least 98% purity by FACS. (B) Purity assessment of isolated BECs, LECs, and other major skin cell types by analysis of marker gene expression. BECs and LECs as well as fibroblasts (FBs), mast cells (MCs), CD14⁺ dermal dendritic cells (DDCs), CD1a⁺ DDCs, epidermal Langerhans cells (LCs), and keratinocytes (KCs), isolated according to the criteria described in “Materials and methods” by FACS, were subjected to RNA isolation. After 2 rounds of linear amplification, cRNAs were hybridized to Affymetrix HG-U133 plus 2.0 GeneChips. Hybridization intensities with probe sets corresponding to cell lineage-associated marker genes (VE-cadherin, PROX-1, collagen 1 [COL1], tryptase, CD14, CD1b, langerin, keratin 14) are given for all cell types (x-axis). If more than one sample was available from a defined cell type, mean values and standard deviations (SDs) are shown.