Transwell migration of peripheral blood leukocytes (PBL).
2017-05-19T17:39:00Z (GMT) by
<p>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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006402#ppat.1006402.s005" target="_blank">S5 Fig</a>). <b>A)</b> 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>B)</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). <b>C)</b> 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).</p>