Efficiency of Lyve-1 CRE-mediated gene deletion and S1P metabolic enzyme expression in LECs. (A) Isolation and identification of LECs. LNs were minced and digested as detailed in Materials and methods. CD31 and gp38 expression allows separation of LECs from other CD45⁻ cells: FRCs, BECs, and double-negative stromal cells (Others). (left plot) LYVE-1 on indicated populations; (right plots) Control stains for each cell type. (B) Flow cytometric analysis of YFP in cells isolated from LNs of Lyve-1 Cre⁺ Rosa26-YFP reporter mice (percentages are shown). The four cell populations are gated according to the scheme in A. Peripheral LNs (pLN; axillary, brachial, and inguinal nodes) and mesenteric LNs (mLN) are shown. The shaded histogram represents Cre-negative cells. (C) Immunofluorescence analysis of YFP in LNs of Lyve-1 Cre⁺ or Rosa26-YFP mice that had been reconstituted with wild-type BM. Fixed LN sections were stained as indicated. Data in A–C are representative of at least three experiments with one to two mice of each type per experiment. (D) Quantitative RT-PCR analysis of S1P metabolic genes in cells sorted from a pool of peripheral and mesenteric LNs using the scheme in A and from spleen tissue or splenic B cells. Data are representative of three separate sorts with 10–15 mice in each sort, with each gene expression measured at least twice. Bars represent means.