Anti mouse gm csf pe

The BMDMs were activated as described above. For intracellular staining, brefeldin A (for IL-12p40/70 and IL-6 detection) or monensin (for IL-10 and GM-CSF detection) was added at the beginning of the activation by LPS. The cells were pretreated with Fc blocker (BD Bioscience), followed by surface staining and fixation/permeabilization using a BD Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions. Anti-mouse F4/80 Alexa fluor 488 (BioLegend), anti-mouse IL-12p40/70-PE (BD Bioscience and BioLgend), anti-mouse IL10-APC (BioLegend) anti-mouse CD206-PE (BioLegend), anti-mouse GM-CSF-PE (BioLegend) were used. Anti-mouse Notch 1-PE (clone N1A) was used to stain for Notch1 using the FoxP3 staining buffer set (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. In some experiments, anti-mouse CD80, anti-mouse CD86, anti-mouse MHCII (BD Pharmingen) and anti-mouse PD-L1 (BioLegend), anti-mouse CD4 (BD Bioscience) antibodies were used for cell surface staining. The cells were acquired on a FACS LSR II (Becton Dickinson) or Cytomics FC 500 MPL (Beckman Coulter) and analyzed with FlowJo software (FlowJo LLC., CA, USA).