Clone avas 12a1

Microbubbles were prepared in-house using previously described methods (Klibanov et al. 1999 ; Klibanov et al. 2006 ). Briefly, biotinylated MBs were formed by sonicating decafluorobutane gas (F2 Chemicals, Lancashire, UK) with a lipid micellar mixture of distearoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL, USA), polyethylene glycol stearate (Stepan Kessco, Elwood, IL, USA) and biotin-PEG3400-distearoylphosphatidylethanolamine (PEG-DSPE, Shearwater Polymers, Huntsville, AL, USA) in normal saline. After MBs were counted using a Coulter Multisizer 3 (Beckman Coulter, Brea, CA, USA) streptavidin (Anaspec Inc, Fremont, CA, USA) was added as a linking molecule at a concentration of 3 µg/10 million microbubbles (Lindner et al. 2001 ).
MBs were divided into two groups for conjugation to two types of antibodies. The first group was conjugated to biotinylated anti-mouse vascular endothelial growth factor receptor 2 (VEGFR2) antibody (clone Avas 12a1, eBioscience, San Diego, CA, USA) (Lee et al. 2008 ; Pochon et al. 2010 ; Willmann et al. 2008c (link); Willmann et al. 2008a (link)) and the second group was conjugated to biotinylated isotype control antibody (clone R35–95, BD Pharmingen, San Diego, CA, USA). All MBs were conjugated within 48 hours before use. Prior to each experiment, MB concentration was measured using a Coulter Multisizer 3.

Microbubbles were prepared using previously described methods (Klibanov et al. 1999 ; Klibanov et al. 2006 (link)). Briefly, decafluorobutane gas (F2 Chemicals, Lancashire, UK) was sonicated with a lipid micellar mixture of distearoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL, USA), biotin-PEG3400-distearoylphosphatidylethanolamine (PEG-DSPE, Shearwater Polymers, Huntsville, AL, USA) and polyethylene glycol stearate (Stepan Kessco, Elwood, IL, USA) in normal saline. A Coulter Multisizer 3 (Beckman Coulter, Brea, CA, USA) was used to count MBs before adding a streptavidin (Anaspec Inc, Fremont, CA, USA) linker at a concentration of 3 μg/10 million MBs (Lindner et al. 2001 ). MBs were conjugated to targeting antibodies within 48 hours before experimentation. To target angiogenic sites on the tumor vasculature, MBs were conjugated to biotinylated anti-mouse vascular endothelial growth factor receptor 2 (VEGFR2) antibody (clone Avas 12a1, eBioscience, San Diego, CA, USA) (Lee et al. 2008 ; Pochon et al. 2010 ; Willmann et al. 2008b (link)). A control group of MBs was conjugated to biotinylated isotype control antibody (clone R35–95, BD Pharmingen, San Diego, CA, USA). Prior to each use, a final measurement of MB concentrations was obtained. MB size ranged between 1 and 3 μm with a typical MB diameter of approximately 2 μm.