Secretion of amphoterin from mononuclear cells. (A-D) Freshly isolated peripheral blood leukocytes were double-immunostained in suspension (A-B) and after adhesion and culture on coverslips for 18 hours in the presence of 10% autologous serum (C-D). Staining for CD14 is shown in panels A and C and for amphoterin in panels B and D. Cells were fixed with 2% paraformaldehyde for 15 minutes and double-stained with anti-CD14 and TRITC-conjugated antimouse immunoglobulins followed by anti-recAtn and FITC-conjugated antichicken immunoglobulins. (E-G) Embolectomy sample taken within 24 hours of the onset of symptoms for lower-limb arterial occlusion was snap-frozen in liquid nitrogen. Frozen sections were fixed with cold acetone, incubated with anti-recAtn (E-F) or nonspecific chicken IgY (G), and detected with peroxidase-labeled antichicken IgY. Scale bar 20 μm (A-G). (H) Secretion of amphoterin from RAW 264.7 cells is induced by IFN-γ and PMA and inhibited with ABC-1 inhibitors DIDS and glyburide. Cells were treated with 20 ng/mL IFN-γ or 10 nM PMA, and medium samples (0, 7, or 10 hours) were collected. Medium (1.5 mL) was concentrated to 15 L and analyzed in anti-recAtn Western blotting. Amphoterin was detected from activated cell culture medium after 7 or 10 hours of culture. Secretion was dose dependently inhibited with DIDS and to a lower extent with glyburide. BSP did not inhibit secretion. Medium samples were analyzed for activity (in mU/mL) of LDH by enzyme activity measurement. The results shown are from a representative experiment of at least 3 experiments. IFN indicates IFN-γ. (I) Optical density (OD) of the amphoterin band in Western blotting of IFN-γ activated (7 hours) cell culture supernatants, and lactate dehydrogenase activity in supernatants was measured. OD of bands from noninhibited samples was defined as 1 (± SD, n = 3).