Anti ly 6g clone rb6 8c5

Mice were injected with 10 µl of anti-Ly6G (clone RB6-8C5) conjugated with PE (Ebioscience, San Diego, CA), at a concentration of 0.2 mg/ml. This concentration of a single bolus injection of antibody is sufficient to allow us to image neutrophils for up to 4 hours without excessive bleaching of signal while in no way affecting neutrophil recruitment parameters [48] (link). In experiments where propidium iodide was used, mice were injected either with 10 µl anti-Ly6G (clone RB6-8C5) conjugated with Alexa680 (Ebioscience, San Diego, CA) at a concentration of 0.2 mg/ml, or 10 µl of anti-Ly6g (clone 1A8) conjugated with PE (BioLegend, San Diego, CA) at a concentration of 0.2 mg/ml. The vasculature was also labelled with 10 µl of CD31 (Clone 390, Ebioscience, San Diego, CA) conjugated to Alexa647 (Molecular Probes, Eugene, OR), at 1.0 mg/ml. CD31 was conjugated in house to Alexa647 with a labeling kit (Molecular Probes, Eugene, OR).

Antibodies used were as follows: anti-Ly-6C (clone HK1.4, eBioscience), anti-Ly-6G (clone RB6-8C5, eBioscience), anti-F4/80 (clone BM8, BD Biosciences), anti-arginase I (Polyclonal sheep IgG, R&D). Cells were washed with PBS, the fixation step was performed with 2% formaldehyde in PBS and the permeabilisation step with 0.5% saponin in PBS. Analysis was performed on a LSRII (BD Bioscience) and results were analyzed using Summit v4.3 software.
The percentages of positive cells with the isotype controls were <1%.