Transwell migration of peripheral blood leukocytes (PBL).

Mononuclear cells and granulocytes were isolated from blood, pooled (i.e. PBL) and incubated in a transwell system for 2 h with explant conditioned medium (ECM) from donor-matched ectocervical explants incubated with culture medium (CM-ECM) or seminal plasma (SP-ECM). Transmigrated cells were immunophenotyped and enumerated by flow cytometry (see S5 Fig). A) Fraction of transmigrated cells out of total number of PBL loaded into a transwell insert for each analyzed cell population (input). PBL were incubated with CM-ECM (white), SP-ECM (red), and with medium only (grey) and medium supplemented with FBS 10% (yellow) as negative and positive controls respectively. Bars represent mean with s.e.m (n = 6). B) N-fold change in the fraction of transmigrated PBL incubated with SP-ECM compared to that of donor-matched CM-ECM. Bars indicate median values. p<0.05 denotes a significant difference with CM (Wilcoxon signed rank test). C) Expression levels of the chemokine receptor CCR5 on transmigrated monocytes. Top, peaks represent PBL untreated cultured (ctrl, gray), cultured with CM-ECM (CM, white), cultured with SP-ECM (SP, red), and unstained control (black) from one representative experiment. Middle, CCR5 mean fluorescence intensity (MFI). Bars indicate median values. Bottom, n-fold change in CCR5 MFI on PBL cultured with ECM compared to untreated cultured PBL (ctrl). Lines connect measurements obtained from donor-matched ECM. p<0.05 denotes a significant difference with ctrl (Wilcoxon signed rank test).