Flow Cytometry analysis of human peripheral blood cells labeling HLA DR with ab136320, followed by a Goat anti-mouse-APC secondary.

Human peripheral blood lymphocytes stained with ab136320 (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 minutes at 22°C. Red blood cells were then lyzed by the addition of Triton X-100 (final concentration - 0.1%) for 15 minutes at 37°C. For experimentation, cells were treated with 50% methanol (-20°C) for 15 minutes at 4°C. Cells were then incubated with the antibody (ab136320, 0.1μg/1x10⁶ cells) for 30 minutes at 4°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 minutes at 4°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 0.1μg/1x10⁶cells) used under the same conditions. Unlabeled sample (blue line) was also used as a control. Acquisition of >30,000 total events were collected using a solid-state 25mW red diode laser (635nm) and 675/30 bandpass filter. Gating strategy - peripheral blood lymphocytes.