48 well boyden chamber

The migration of primary human monocytes and B cells isolated from buffy coats, or of murine preB 300.19 cells stably expressing the human chemokine receptors CCR2^{75 (link)}, CCR6, CXCR1^{76 (link)} and CXCR6^{77 (link)} was assayed using 48-well Boyden chambers (Neuro Probe, Cabin John, MD) with polyvinylpyrrolidone-free polycarbonate membranes with pore size of 3 μm for primary human B cells and 5 μm for the other cell types, as previously described ^{78 (link)}. Briefly, 10⁵ primary human B cells or 5x10⁴ primary human monocytes and murine preB 300.19 cells were diluted in RPMI-1640 supplemented with 20 mM Hepes, pH7.4, and 1% pasteurized plasma protein solution (5% PPL SRK; Swiss Red Cross Laboratory, Bern, Switzerland). Cells were then added to the upper wells and the chemokine (with or without antibodies) to the bottom wells. After 120 min of incubation for primary human B cells and 90 min for the other cell types, the membrane was removed, washed on the upper side with phosphate-buffered saline (PBS), fixed, and stained with DiffQuik. All assays were done in triplicate, and for each well the migrated cells were counted at 100-fold magnification in 5 randomly selected high-power fields (5HPF).

Chemotaxis assays were performed in 48-well Boyden chambers (Neuro Probe, Gaithersburg, MD, USA). EoL-1 and Jurkat cells were washed and resuspended in chemotaxis medium containing 1% BSA and 2 mM HEPES. Assays were performed in triplicates using 2×10⁵ cells/well with a 5-µm-pore size PVPF membrane (Osmonics, Minnetonka, MN, USA). A 27 µl aliquot of indicated concentrations of eotaxin was added to the lower chamber. A filter was overlaid and then 50 µl of the cell suspension was added to the upper chambers. The chamber was incubated at 37℃ in 5% CO₂ for 3 h. Then, cells which migrated to the lower chamber were counted. Chemotaxis was expressed as a chemotaxis index.

Invasion assays were performed using 48-well Boyden chambers (Neuroprobe, Gaithersburg, MD, USA). The lower wells of the chamber were filled with a standard culture medium. The chamber was assembled using polycarbonate filters (Neuroprobe) coated with Matrigel. Cells in a serum-free medium (5×10⁴ cells/well) were seeded in the upper compartment of the chamber. After incubation for 24 h, cell migration was quantified by counting the number of migrated cells after staining with hematoxylin and eosin under a light microscope (Olympus, CKX31-11 PHP, Tokyo, Japan). The data shown are the mean values of three independent experiments.

KC migration was assayed quantitatively using a 48-well Boyden chamber (Neuro Probe, Tokyo, Japan), as described in our previous report (Tokumaru et al., 2005 (link)). To investigate the involvement of nuclear IL-33 or STAT3 activation in HB-EGF−induced KC migration, siRNA-transfected cells or Ax-infected cells were collected and added into the upper wells of the chamber, whereas HB-EGF together with or without specific inhibitors were added into the bottom wells of the chamber.