Flow cytometry analysis of Chicken anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647 (A21463) was performed using K-562 cells stained with alpha Tubulin Mouse Monoclonal Antibody (A11126). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with alpha Tubulin antibody or with mouse isotype control at 3-5 µg/million cells in 2.5% BSA and incubated for 2 hours at room temperature. The cells were then labeled with Chicken anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647 (A21463) at a dilution of 1:500 for 1 hour at room temperature. A representative 10,000 cells were acquired and analyzed for each sample using the Attune® NxT Acoustic Focusing Cytometer. Panels a, b and c represent cells stained with the secondary antibody alone, isotype control and alpha Tubulin Monoclonal Antibody respectively. Median fluorescence intensity from the three samples is compared in panel d.