Human whole blood stained with ab11029 (right) or mouse IgG1κ (left). Red blood cells of 200µl human whole blood were lysed, then cells were incubated for 30 min on ice in 1x PBS containing 10µg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab11029) or mouse IgG1κ isotype (ab170190) (100µl at 10 µg/ml) for 30 min on ice.

The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor ^® 488, pre-adsorbed) (ab150177) was used at 1/2000 dilution for 30 min at 4°C.

Acquisition of >30,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. Events were gated on viable single cells.

Human peripheral blood lymphocytes stained with ab11029. 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 lysed by the addition of Triton X-100 (final concentration - 0.1%) for 15 min at 37°C. For experimentation, cells were stained with anti-CD11c ab11029 (right panel) at 1/100 dilution for 30 min at 4°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-mouse IgG (H&L) (ab150117) at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (left panel) was mouse monoclonal IgG1 (ab170190) used under the same conditions.

Acquisition of >30,000 total events were collected using a 50mW Argon Blue laser (488nm) and 530/30 bandpass filter. Gating strategy – events were collected with the forward and side light-scatter characteristics of viable cells.