Anti-CD11b antibody [M1/70] (ab8878)

ab8878 stained in Raw264.7 cells. Cells were fixed with 100% methanol (5 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab8878 at 1µg/ml overnight at +4°C. The secondary antibodies was ab150165 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

Overlay histogram showing Human monocytes stained with ab8878 (red line). PBMCs were pre-incubated in Human Fc block for 30 mins at 4ºC. Cells were then incubated with the antibody (ab8878, 1µg/1x10⁶ cells) for 30 min at 4ºC. The secondary antibody used was a goat anti-rat Alexa Fluor® 488 (IgG H+L) (ab150165) at 1/2000 dilution for 30 min at 4ºC.

Isotype control antibody (black line) was rat IgG2b [RTK4530] (ab18541, 1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 band pass filter.

Human monocytes were identified with CD14 APC (ab91144) multi-color staining.

Overlay histogram showing Human macrophages stained with ab8878 (blue line). Cells were incubated with human immunoglobulin for 30 min at 4°C to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab8878, 20 µg/mL) for 30 min at 4ºC. The secondary antibody used was a mouse anti-rat FITC (IgG2b gamma chain) (ab99671) at 1 µg/mL for 30 min at 4ºC. Isotype control antibody (black line) was Rat IgG2b [RTK4530] (ab18541) used under the same conditions.

Overlay histogram showing Human macrophages stained with ab8878 (red line). Cells were pre-incubated in Human AB serum (10%) for 20 mins at 4ºC. Cells were then incubated with the antibody (ab8878, 0.1µg/1x10^6 cells) for 30 min at 4ºC. The secondary antibody used was a goat anti-rat Alexa Fluor® 488 (IgG H+L) (ab150165) at 1/2000 dilution for 30 min at 4ºC. Isotype control antibody (black line) was rat IgG2b [RTK4530] (ab18541, 1µg/1x10^6 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.