Transwell device

To evaluate the mechanism for IgA production in vitro, B cells (5×10⁵) were either directly stimulated with LG2055 (10 µg/ml), BAFF (500 ng/ml, Sigma), TGF-β1 (0.001–1 ng/ml, R&D Systems), and LPS (10 µg/mL, InvivoGen) or co-cultured with CD11C⁺ cells derived from PP cells (0.5×10⁵) or BMDC (1×10⁵) in the presence or absence of LG2055 (10 µg/ml), LE135 (500 nM), SB505124 (5 µM), anti-human/mouse TLR2 (5 µg/ml), Pam3CSK4 (1 µg/mL, InvivoGen, CA), and FSL-1 (1 µg/mL, InvivoGen) in 96 well-round bottom plate (BD Bioscience) for 7 days. To block cell-to-cell contact, B cells (2×10⁶) and BMDC (4×10⁵) were cultured in 24 well culture plate (BD Bioscience) with or without a transwell device (BD Bioscience) in the presence or absence of LG2055 (10 µg/ml) for 7 days. The level of IgA in the culture supernatants was measured by mouse IgA ELISA.

Before hUCB-MSC coculture, a tau K18 fragment mixture (with heparin and DTT) was placed in multiwell plates in the presence of buffers. Then, hUCB-MSCs (2 × 10⁴ cells/cm²) were cocultured in the upper chamber of a Transwell device (pore size, 1 mm; BD Biosciences, Franklin Lakes, NJ, USA) in serum-free conditions. The final concentrations of recombinant human GAL-3 and GDF-15 were both 20 ng/mL and that of CD147 was 100 ng/mL.