Anti her 2 antibody

DiI with emission wavelength near 570 nm was chosen as a liposome tracer. This fluorescent color was detected by FL2 channel of flowcytometer. Two membranous macro-molecules (gp63 as a leishmania parasite surface Ag and HER-2 as a SK-BR-3 cell line surface molecule) were selected for tracking PMVs. Anti-gp63 antibody (Serotech), anti-HER-2 antibody or Herceptin (Roche) were used as primary antibodies. Mouse FITC-conjugated anti-rat IgG and FITC-conjugated anti-human IgG (Sigma chemical Co., St. Louis, USA) were used as secondary antibodies. This fluorescent color was detected by FL1 channel of flowcytometer. To determine the percentage of fused particles which are stained with both DiI and FITC, 100 µl of the final materials derived from fusion reactions were washed twice with PBS by centrifugation at 4000 g for 10 min. Pellets were resuspended in 100 µl anti-gp63 antibody (1/100 dilution) or anti-HER2 antibody (1/20000 dilution) and incubated for 30 min at room temperature. Tubes were washed twice with PBS and FITC-conjugated Abs were added (1/50 dilution) for 30 min at room temperature. The tubes were washed again three times with PBS and the pellet was resuspended in 0.5-1 ml PBS. Flowcytometric evaluation of these samples was carried out by BD flowcytometer (Becton Dickinson, US) equipped with Cell Quest pro software and data were analyzed using WinMDI software.

After staining of DiI contained fused particles with anti-gp63 antibody (Serotech) or anti-HER-2 antibody (Roche), fused particles were also examined by fluorescent microscope (Olympus IX 81, Japan). Band-pass filters for FITC and DiI were ex-WIB (460-495 nm) and ex-WIG (530-550nm) while emission filters were 510-550 nm and 575-625 nm, respectively.