VCAM-1/Fc binds to murine high-affinity α4 integrins and identifies subtype-specific regulation of integrin affinity. (A) Representative flow cytometry profiles of human IgG, ICAM-1/Fc, and VCAM-1/Fc binding to mouse peripheral blood leukocytes. Binding was carried out in diluted whole blood with 1 mM Ca²⁺ and Mg²⁺ with or without 1 mM Mn²⁺. (B) Blockade of VCAM-1/Fc binding with function-blocking anti-α4 integrin and anti-α4β7 antibodies (30 minutes of pretreatment) in Ca²⁺/Mg²⁺ containing buffers with or without Mn²⁺. Data are expressed as mean ± SEM, n = 4 mice. (C) IgG, ICAM-1/Fc, and VCAM-1/Fc binding to different leukocyte subtypes, identified by immunostaining for specific cell surface markers. MnCl₂ (1 mM) was included in some assays (right panel). Data pooled from C57BL/6 and BALB/c mice are expressed as the mean ± SEM, n = 10 mice. (D) Blockade of VCAM-1/Fc binding to B cells, T cells, and monocytes (Mono) with anti-α4 integrin (mean ± SEM, n = 4 mice).