Immunofluorescent Detection of A3B and γ-H2AX

Myeloma cells were air-dried and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 minutes on glass slides using Shandon cytospin 2 (THERMO FISHER SCIENTIFIC). Fixed cells were permeabilized, reduced and denatured for 30 minutes in PBS buffer containing 0.5% SDS, 5% β-mercaptoethanol and 10% FBS. Then, cells were washed three times with PBS containing 4% FBS and 0.1% Triton X-100 (PFT buffer)^{65 (link)}, and incubated with a purified rabbit anti-A3B antibody for 1 hour. Subsequently, cells were incubated with a goat anti-rabbit IgG (H + L)-Alexa Fluor® 488 preadsorbed antibody (Abcam, ab150081) for 30 min in the dark. γ-H2AX foci analysis was performed as previously described^{29 (link)} using a mouse anti-phospho-histone H2A.X (Ser139) antibody (Millipore, clone JBW301) as primary antibody and a goat anti-mouse IgG (H + L)-Alexa Flour® 594 preadsorbed antibody (Abcam, ab150120) as secondary antibody. All antibodies were diluted with 3% BSA and 0.5% Tween in PBS. Around two hundred cells were observed and scored with a confocal laser scanning microscope (TCS-SP8, Leica) or a fluorescence microscope (BZ-9000, KEYENCE).