Costar transwell chamber

The migration of splenic and BM B cell populations in response to chemokine gradients was performed in 6-well Costar transwell chambers (5 μm pore size, Corning). Briefly, a suspension of 10⁶ mononuclear cells in RPMI 1640 supplemented with 10% FCS was added to each insert in a well containing a solution of chemokine (R&D Systems). In some experiments, blocking anti-CXCR4 MAB21651 (247506, R&D Systems) or anti-CCR7 MAB3477 (4B12, R&D Systems) antibodies were added to the cells prior to migration. Wells containing medium without chemokines were used as controls. After 2 h at 37°C, cells in the bottom of the wells were harvested, diluted in FACS staining buffer and incubated with antibodies and counting beads to discriminate and quantify B cell subsets. The migration of Nalm-6 cells was performed in 96-well Costar transwell chambers (5 μm pore size, Corning NY). A cell suspension of 10⁴ Nalm-6 in RPMI 1640 supplemented with 10% FCS was added to each insert. Wells containing medium without chemokines were used as controls. After 1 h at 37°C, cells in the bottom of the wells were counted by using the ATPlite luminescence assay kit (PerkinElmer, Waltham, MA). The results are expressed as chemotaxis index, i.e., the ratio of cells migrating in response to the chemoattractant over cells migrating toward the medium alone.

Supernatants from THP-1 derived M2 macrophages with or without fucoidan pretreatment (24 h), recombination human CCL22 (100 ng/ml) or blocking mAbs (1 μg/ml) were plated into the bottom of migration chambers of 24-well plate. For tumor cell migration assay, 5 × 10⁴ MHCC-97H cells in 200 μl RPMI 1640 medium containing 0.1% FBS were added to the top of 8 μm-pore Costar Transwell chambers (Corning Life Sciences, Corning, NY, USA). After incubated at 37 °C for 24 h in an atmosphere containing 5% CO₂, the cells on the top side of filter were wiped off with cotton swab, and migrating cells located on the lower surface were fixed in methanol and stained with eosin. For lymphocyte migration assay, 4 × 10⁶ fresh human PBMCs or 2 × 10⁶ isolated CD4⁺ T cells in 200 μl RPMI 1640 medium containing 0.1% FBS were seeded in the top of the 5 μm-pore Costar Transwell chambers (Corning Life Sciences). Following 6 h incubation at 37 °C, cells migrating to the lower chambers were collected and used for flow cytometry.

Invasion assays were performed as previously described using 8-μm-pore-size Costar Transwell chambers (Corning, NY) and Transwell filters coated with a thin layer of BD Matrigel (BD Biosciences)⁵⁰. Prior to invasion assay, cells were pretreated for 30 minutes with inhibitors, including the CXCR4 neutralizing antibody (12G5), isotype control antibody, or AMD3100 (Sigma-Aldrich). Cells (2 × 10⁴) were suspended in 200 μl of serum-free RPMI-1640 medium placed in the upper chamber; serum-free RPMI-1640 medium containing 30 ng/ml CXCL12 was placed in the lower chamber. Cells were incubated for 18 hours and then removed from the upper surface of the filter by scraping with a cotton swab. The cells that adhered to the bottom of the membrane were stained with Giemsa solution (Sigma-Aldrich).