Soluble icam 1 vcam 1 conjugated to human fc

Splenocytes from immunized mice or LPS-differentiated cells (2 × 10⁶ per 100 μl of RPMI containing 1 mM MgCl₂ and 1 mMCaCl₂) were incubated with soluble ICAM-1/VCAM-1 conjugated to human Fc (20 μg ml⁻¹; R&D Systems) for 30 min at room temperature, with or without inhibitors to Src family tyrosine kinases (PP2,10 μM) or PI3-kinase (LY294002,10 μM). Binding of ICAM-1/VCAM-1 was detected by incubating cells with APC-conjugated anti-human Fc (BioLegend) for 20 min at 4 °C, followed by dual staining with anti-CD138 and anti-B220 for 20 min at 4 °C to identify ASCs by flow cytometry. Antibodies were diluted (1:100) in PBS containing 5% fetal bovine serum.