Blank control: Raji (blue). Primary Antibody: Rabbit Anti-ENPP3 antibody(bs-1568R), Dilution: 1μg in 100 μL 1X PBS containing 0.5% BSA; Isotype Control Antibody: Rabbit IgG(orange),used under the same conditions ); Secondary Antibody: Goat anti-rabbit IgG-PE(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA. Protocol The cells were fixed with 2% paraformaldehyde (10 min) , then permeabilized with 90% ice-cold methanol for 30 min on ice. Primary antibody (bs-1568R, 1μg /1x10^6 cells) were incubated for 30 min on the ice, followed by 1 X PBS containing 0.5% BSA + 1 0% goat serum (15 min) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/PE antibody was added into the blocking buffer mentioned above to react with the primary antibody at 1/200 dilution for 30 min on ice. Acquisition of 20,000 events was performed.

Black line : Positive blank control (Raji); Negative blank control (Molt4） Green line : Primary Antibody (Rabbit Anti-ENPP3 antibody (bs-1568R) ) Orange line：Isotype Control Antibody (Rabbit IgG) . Blue line : Secondary Antibody (Goat anti-rabbit IgG-PE)/Goat anti-rabbit IgG-AF488) Raji（Positive）and Molt4（Negative control）cells (black) were incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with ENPP3 Antibody(bs-1568R)at 1:50 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2% BSA in PBS, followed by secondary antibody(blue) incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).