Af 488 conjugated goat anti mouse antibody

Binding specificity of the mouse anti-human CD44 (IgG1κ, clone 515) antibodies, which were used for the SR imaging experiments, to CD44 on KG1a cells was evaluated by flow cytometry. KG1a cells (10⁶ cells ml⁻¹) were incubated with either anti-human CD44 antibody (10 μg ml⁻¹) or purified mouse IgG1κ isotype (10 μg ml⁻¹; BioLegend) in HBSS at 4^°C for 30 min. Subsequently, the KG1a cells were incubated with AF-488–conjugated goat anti-mouse antibody (5 μg ml⁻¹, IgG, Invitrogen) in HBSS at 4°C for 20 min. For the secondary antibody control, KG1a cells (10⁶ cells ml⁻¹) were incubated with AF-488–conjugated goat anti-mouse antibody (5 μg ml⁻¹) in HBSS at 4°C for 20 min. To assess the expression level of CD44 on the surface of KG1a cells, we fixed CytD-treated cells, MβCD-treated cells, and control cells with 1% paraformaldehyde in HBSS for 20 min at room temperature and incubated them with either mouse anti-human CD44 antibody (10 μg ml⁻¹; IgG1κ, clone 515) in HBSS at 4°C for 30 min. The cells were then incubated with AF-488–conjugated goat anti-mouse antibody (5 μg ml⁻¹) in HBSS at 4°C for 20 min. The fluorescence intensity was determined using a FACSCanto flow cytometer (Beckman Dickenson).