Anti-CD3 epsilon antibody [EP449E] (ab52959)

Human peripheral blood lymphocytes stained with ab52959 (red line). Human whole blood was processed using a modified protocol based on Chow et al, 2005 (PMID: 16080188). In brief, human whole blood was fixed in 4% formaldehyde (methanol-free) for 10 min at 22°C. Red blood cells were then lyzed by the addition of Triton X-100 (final concentration - 0.1%) for 15 min at 37°C. For experimentation, cells were treated with 50% methanol (-20°C) for 15 min at 4°C. Cells were then incubated with the antibody (ab52959, 1/1000 dilution) for 30 min at 4°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >30,000 total events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. Gating strategy - peripheral blood lymphocytes.