Transwell chamber filters

Transwell chamber filters (Corning, NY, USA, Cat. 3422) were coated with Matrigel (BD, NY, USA, Cat. 356234). After infection with lentivirus, LM3 or SK-HEP-1 cells were suspended in serum-free DMEM or MEM media, and then, 2 × 10⁴ cells were seeded into the upper chamber in a volume of 300 μL. The chamber was cultured in a well containing 500 μL of DMEM or MEM media with 10% FBS at 37 °C for 18 h. Cells on the upper side of the membrane were removed using cotton swabs and those on the underside were stained and counted. Four high-powered fields were counted for each membrane.

Transwell chamber filters (5 µM, Corning) or 24-well plates (control) were coated with an attachment factor (Gibco) for 1 h at 37°C. The solution was then removed and the filters were dried. These filters were seeded with 2.5 × 10⁴ endothelial MLEC cells (42 (link)) per filter in 200 µl of complete DMEM. Two wells out of a 24-well plate were used to monitor the confluency of MLEC cells and the integrity of the endothelial barrier. The cells were incubated for 2 days at 37°C. When the cells reached confluency, the monolayers were treated with 5 nM TNF-α overnight. After incubation, the filters were washed with DMEM and the top and the bottom chambers were filled with 50 and 600 µl DMEM (without FBS), respectively. CXCL13 (1 µg/µl) was added in some of the bottom chambers. In some cases, CFSE-labeled resting or activated B cells were pretreated with α-CD44 clone IM7 for 1 h or left untreated. Then, 4 × 10⁵ B cells were added on top of each filter and incubated for 4 h. The filters were then carefully removed and the cells at the top and the bottom of each well were stained with α-B220-PB and analyzed by flow cytometry.