Qifikit calibration beads

MPIO (10 μg) or USPIO (70 μg) were diluted in 200 of PBS buffer, and 2 μL of Alexa Fluor 647 goat anti-rat IgG (H+L) (Cat. No. A21091, 5 fluorophores per antibody, Invitrogen) was added. For MPIO, samples were shaken for 30 min, pelleted using a magnet, washed twice with PBS/0.01% Tween, and resuspended in 400 μL of PBS. Qifikit calibration beads (Dako, Agilent Technologies, Stockport, UK), used as a reference, were prepared according to the manufacturer’s protocol, but substituting the provided fluorescently-conjugated antibody by Alexa Fluor 647 goat anti-mouse IgG (H+L) (Cat. No. A21050, 5 fluorophores per antibody, Invitrogen). Flow cytometry experiments were performed on a BD FACS-Calibur flow cytometer, using channel FL4 (661/16 nm band pass filter). Calibration curve was performed, according to manufacturer protocol, by linear regression of the log–log plot of antibody loading versus fluorescence intensity. For USPIO, sample was centrifuged at 186,000 g for 1.5 h, the supernatant discarded, and the pellet reconstituted in 1 mL of PBST. The sample was pelleted again (186,000 g for 1.5 h) and reconstituted in 200 μL of PBS. Fluorescence intensity of the samples was measured and compared to a calibration curve composed of serial dilutions of Alexa Fluor 647 goat anti-rat IgG (H+L). Results are reported as mean ± SD.