Expression and secretion of LN-8 by isolated blood neutrophils. (A) Detection of laminin α4, β1, and γ1 chains in neutrophils by immunofluorescence flow cytometry. Reactivity of mAbs with nonpermeabilized and permeabilized cells is shown. Mouse IgG (dotted line) and mAbs (shaded peak) C3D-1 (CD15), MPO-7 (myeloperoxidase), 5D8 (LNα4), 2G6 (LNβ1), and CAF-2 (LNγ1) were used. CD15 and myeloperoxidase were used as neutrophil and cell permeabilization markers, respectively. (B) Western blot analysis of laminin β1 mAb (DG10) immunoaffinity-purified protein from neutrophil lysate (top panel) and neutrophil secretion (bottom panel). Secreted material as supernatant was obtained after stimulating neutrophils with 200 nM TPA for 20 minutes. mIgG (negative control), mAb DG10 (LNβ1), mAb 22 (LNγ1), and mAb 6C3 (LNα4) were used. Bands of 230, 220, and 180 kDa, corresponding to LNβ1, LNγ1, and LNα4 chains, respectively, were detected.