1 channels at the rear part of cells induces localized cell shrinkage and retraction of this cell part thereby promoting cell migration [9]. Moreover, the migratory activity of macrophages infiltrating atherosclerotic lesions and exhibiting an enhanced KCa3.1-expression was sensitive to the blockade of KCa3.1 [10]. Recently, it has been shown that KCa3.1 is also involved in the migration of lung DCs towards CCL19 or CCL21 using a transwell Selleck GS-1101 system [11]. We here explored the role of KCa3.1 channels in LPS-induced DC migration. Additionally, cell volume changes of DCs upon stimulation with LPS were monitored since cell swelling has been described as a crucial event for cell migration

in leukocytes and DCs [12, 13]. BMDCs were obtained from 8- to 12-week-old female C57BL/6 N

(Charles River, Sulzfeld, Germany), TLR4−/− mice (on the C57BL/6 background), KCa3.1−/− mice (on the C57BL/6 background) as previously described [14]. KCa3.1-deficient mice (KCa3.1−/−) were generated check details as described [15]. TLR4−/− mice [3] were kindly provided by Tilo Biedermann (Department of Dermatology, University of Tübingen). Briefly, immature BMDCs were generated from bone marrow-derived cells by cultivating them in RPMI 1640 medium (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (Sigma, Taufkirchen, Germany), 2 mM L-glutamine (Invitrogen, Darmstadt, Germany), 100 U/mL penicillin, 100 µg/mL streptomycin, 1% (vol/vol) nonessential amino acids, 1 mM sodium pyruvate (all from Biochrom), 50 µM β-mercaptoethanol (Sigma), and 200 U of GM-CSF/mL produced by mouse myeloma cells P3 × 63. On Day 8 of culturing BMDCs were seeded in uncoated 6-well plates (Greiner Bio-One, Frickenhausen, Germany) at a density of 1 × 106 cells in supplemented RPMI 1640 medium and stimulated or not with 500 ng/mL LPS (ultra pure, from Salmonella minnesota) (Calbiochem 437628, Darmstadt, Germany) up to 4 hr. At the indicated time points, 1.25 × 105 cells were harvested and analyzed by

flow cytometry. As a measure of cell size the mean of the forward scatter of BMDCs were analyzed by flow cytometry on a FACSCalibur (BD Biosciences, Heidelberg, Amobarbital Germany) using WinMDI version 2.8 software (J. Trotter, The Scripps Institute, La Jolla, CA). As a control, aqua bidest (20%) to induce oncotic cell swelling, and staurosporine (4 µM, Sigma) to induce cell shrinkage, respectively, were added to the cell culture medium. On Day 8 of culturing 5 × 105 BMDCs in supplemented RPMI 1640 medium were seeded per insert of a BD Falcon™ FluoroBlok™ 24-Multiwell Insert System (Heidelberg, Germany) containing a membrane with 6.5 mm diameter and 3 µm pore size. The bottom wells of this transwell system were filled with supplemented RPMI medium with or without 100 ng/mL CCL21 (PeproTech, Hamburg, Germany), a chemoattractant and ligand for CCR7.