Permeable polycarbonate membrane inserts

B cells were purified from the spleens of C57BL/6J mice by negative selection (B cell Isolation Kit, mouse, Miltenyi Biotec). For the migration assay, primary mouse airways were isolated 1 day prior and treated with 10% CSE in airway epithelial cell culture medium (PromoCell) or culture medium alone for 24 h. To inhibit CYP7B1, clotrimazole in DMSO was diluted with culture medium or combined with 10% CSE to a final concentration of 1 μM. The supernatants were transferred as conditioned medium to the lower well of 24‐well transwell plates (Permeable Polycarbonate Membrane Inserts, Corning, Fisher Scientific), for inducing B‐cell migration, while the airway samples were snap‐frozen in liquid nitrogen for RNA isolation. Freshly isolated B cells at 2.5 × 10⁶/ml in 100 μl were activated by unconjugated AffiniPure F(ab’)₂ Fragment Goat anti‐mouse IgM, μchain‐specific antibody (1.3 μg/ml, 115‐006‐020, Jackson Immunoresearch Laboratories) for 1 h at 37°C in 5.0 μm pore‐sized transwell inserts (Permeable Polycarbonate Membrane Inserts, Corning, Fisher Scientific). Transwell inserts were then placed into the wells of conditioned medium and incubated for 3 h at 37°C. Migrated B cells were collected and counted by Neubauer Chamber and reported as percentage of input.

To analyze cell proliferation, 2–4 × 10³ cells were seeded onto a 96-well culture plate with 100 μL conditioned medium (CM) and collected for sulforhodamine B (SRB) assay after indicated time periods. The cells were fixed with 10% trichloroacetic acid (TCA), washed with distilled water, and stained with sulforhodamine B (SRB; Sigma) in 1% acetic acid. SRB left on the cells was dissolved using 10 mM Tris-HCl, and optical density was measured at 560 nm by using a microplate reader (Bio-Rad).
To perform transwell migration assay, plates with permeable polycarbonate membrane inserts (8 μm pore-size, Corning, NY, USA) were used. The upper and lower chambers were plated with BMDM or THP-1 cells and 4T1 or MDA-MB-231 cells respectively. CCL2 neutralizing antibody and control antibody were added into both sides of the insert at 5 μg/mL. After 12–24 h, plates were fixed with 4% (w/v) paraformaldehyde and cell migration was assessed based on the number of cells that migrated through the insert by crystal violet staining (3 different randomly selected 10× fields in each insert).