Anti gr 1 apc clone rb6 8c5

MDMs were stained with anti-human CD11b-APC (clone M1/70, Biolegend), washed in PBS/0.5% BSA, fixed and permeabilized using the Perm/Fix kit (Nordic MUbio) according to manufacturer’s instructions. Cells were incubated with the anti-human p47^phox antibody (clone 1, Becton Dickinson) in permeabilization buffer for 20 min followed by staining with a secondary FITC-conjugated mouse IgG (Beckman Coulter) before analysis in the LSRII instrument (BD Bioscience).
Murine cells (1 × 10⁶ cells) were initially co-stained with a mix of anti Gr-1-APC (clone RB6-8C5, Biolegend) and anti CD11b-PercpCy5.5 (clone M1/70, Bioscience), or with a mix of anti B220-APC (clone RA3-6B2, Bioscience) and anti-CD3-PeCy5 (clone 17A2, Biolegend) in the presence of γFc blocking and then intracellularly stained for p47^phox as described above. PLB985 clones were analysed for p47^phox expression after granulocytic differentiation by staining with an anti-human CD11b-PE (clone M1/70, Biolegend) followed by intracellular staining with anti-human p47^phox-(clone 1) APC labelled by Beckton Dickinson services. FACS analysis was performed in CyAn^TM ADP (Beckman Coulter).

Dissected tumours were dissociated to obtain single-cell suspensions for antibody staining and immune cell assessment. Prior to the antibody staining, dead cells were labelled with the Fixable Viability Due eFluor™ 780 (eBiosciences, San Diego, CA, USA). The following anti-mouse antibodies were used for flow cytometry: anti-CD45-FITC (clone 30-F11, Biolegend, San Diego, CA, USA), anti-CD3-BUV395 (clone 145-2C11, BD), anti-CD4-BV421 (clone GK1.5, Biolegend), anti-CD8-PE-eFluor610 (clone 53-6.7, eBiosciences), anti-Foxp3-PE (clone FJK-16S, eBiosciences), anti-CD11b-PE-Cy7 (clone M1/70, Biolegend), anti-F4/80-BV510 (clone BM8, Biolegend), and anti-Gr1-APC (clone RB6-8C5, Biolegend).