Immunofluorescence staining. RMS cells were first fixed with 3.7% formaldehyde solution to perform immunofluorescence staining with anti-CD47 or anti-calreticulin mAbs. Additionally, several sections from frozen specimens of RMS tumors were analyzed. Cells and tissue slides were fixed for 5 min in a 1:1 acetone and methanol mixture (Merck) and washed in PBST (PBS with 0.1% Tween 20, Sigma-Aldrich). Blocking was performed using 1% normal goat serum (Dako Cytomation) for 30 min followed by primary antibody diluted in PBST to 10 mg/ml. Slides were washed three times for 5 min in PBST. Secondary Alexa 546-conjugated goat anti-mouse antibody (1:500; Molecular Probes/Invitrogen) was added for 30 min at room temperature, followed by three times washing for 5 min. For counterstaining of the nucleus, Hoechst 33342 was used (1:2000; Sigma-Aldrich). Slides were mounted with fluorescence mounting medium (Dako Cytomation) and subsequently analyzed by fluorescence microscopy (AxioVision; Carl Zeiss). Cell proliferation. RMS cells (1 104) were seeded out in a 96-well plate. After incubation over night, serial 10-fold dilutions of doxorubicin (Cell Pharm) ranging from 100 to 0.00001 mg/ml were added (as shown in Fig. 5). Cell viability was assessed 96 h later by an MTT assay. Therefore, 25 ml of 12 mM MTT [3-(4,5- dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide] (Sigma- Aldrich) dissolved in PBS were added to each well containing 100 ml cell suspension. Incubation was stopped 3 h later with 100 ml of 10% SDS in acid water (Merck). Absorption was measured at 570 nm after incubation over night using a Milenia Kinetic Analyzer (DPC Biermann). All assays were performed in triplicates. The percentage of viability was calculated by normalization between background of cultures without cells and untreated cultures as control experiments. Generation of macrophages. Peripheral blood mononuclear cells (PBMCs) isolated from buffy coats were plated into 25 ml cell culture flasks in serum-free X-VIVO 20 medium (Lonza). After 1 h of incubation at 37°C, non-adherent cells were removed by washing twice in PBS. Adherent cells were then incubated in RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% L-glutamine, 1% non essential amino acids, and 1% sodium pyruvate (Biochrom) for 6 d with medium changes every 48 h. On day 6, 50 U/ml GM-CSF (BD Biosciences) were added to activate differentiated macrophages. On day 7, cells were incubated for 5 min in PBS, scraped off with a sterile cell scraper, collected by centrifugation, and re-suspended in culture medium. Phagocytic activity of macrophages. RMS (2 104) cells were seeded out on a 96-well plate and incubated with 10 mg/ml anti- CD47 mAb for 30 min. Afterwards, macrophages were added at an effector to target (E:T) ratio of 2.5:1. Digital images were taken at a 6.3-fold magnification with AxioCam MRc (Carl Zeiss) on a Leitz Fluovert FS optical microscope (Leica) 24 and 96 h later. The cell free area in the cell layer was measured using an image analyzer system (AxioVision 3.1; Carl Zeiss). RMS cells (1 104) were seeded out on a 96-well plate and incubated with either 0.01 mg/ml or 0.1 mg/ml doxorubicin (Cell Pharm) for 24 h. Cells were incubated for 30 min with 10 mg/ml anti-CD47 mAb, as indicated. A blocking peptide to calreticulin (MBL) at 4 mg/ml was added to the tumor cells.19 Subsequently, macrophages were added at an E:T ratio of 2:1. Target phagocytosis was monitored after 24 h using MTT assay as described previously. 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