Cd163 apc vio770

On day 6 primed cells were harvested using ice cold PBS containing 5 mM EDTA. Cells were washed with PBS and staining was performed for 30 min on ice with surface markers for CD80-APC (Biolegend, #305220), CD86-VioBright 515 (Miltenyi Biotec, #130-116-165), CD163-APC-Vio770 (Miltenyi Biotec, #130-112-131), CD206 (Biolegend, #321110) or NLRP3 (Miltenyi Biotec, # 130-111-209) following manufacturer’s instructions. Intracellular staining of p-mTOR and HIF1α was performed after 4% formaldehyde fixation. Cells were washed once with permeabilization buffer (Biolegend, #421002) and stained with PE-Cyanine7 anti-human p-mTOR (Ser2448) (eBioscience, #25-9718-41) or PE anti-human HIF1α Antibody (Biolegend, #359704) antibodies for 20 minutes in the dark at room temperature. PE mouse IgG2b κ (Biolegend) and PE-Cyanine7mouse IgG2a κ (eBioscience^TM) were used as isotype control for intracellular staining. After staining the cells were washed with PBS and FACS analysis was performed using Guava easyCyte (Millipore).

On day 1, day 3, and day 6 primed cells were harvested using ice cold PBS containing 5 mM EDTA. Cells were washed with PBS and staining was performed for 30 min on ice with surface markers for CD80-APC (Biolegend, #305220), CD86-VioBright 515 (Miltenyi Biotec, #130-116-165), CD163-APC-Vio770 (Miltenyi Biotec, #130-112-131) and CD206 (Biolegend, #321110) and following manufacturer's instructions. Intracellular staining of p-mTOR and HIF1α was performed after 4% formaldehyde fixation. Cells were washed once with permeabilization buffer (Biolegend, #421002) and stained with PE-Cyanine7 anti-human p-mTOR (Ser2448) (eBioscience, #25-9718-41) or PE anti-human HIF1α Antibody (Biolegend, #359704) antibodies for 20 min in the dark at room temperature. PE mouse IgG2b κ (Biolegend) and PE-Cyanine7 mouse IgG2a κ (eBioscience™) were used as isotype control for intracellular staining. After staining the cells were washed with PBS and FACS analysis was performed using Guava easyCyte (Millipore).