Allophycocyaninconjugated anti mouse cd11b

For in vivo staining, tumor tissue was minced and digested with trypsin for 20 minutes at 37°C. Tissue homogenate was then filtered through a 40 μm filter and prepped using Fixation/Permeabilization solution according to the manufacturer's instructions (BD Pharmingen. San Diego, CA). Multiple-color FACS analyses was performed at City of Hope FACS facility using a 3-laser CyAn immunocytometry system (Dako Cytomation, Fort Collins, CO), and data was analyzed using FlowJo software (TreeStar, San Carlos, CA) as described before (19 (link)). PerCP-conjugated antibodies to mouse CD45 (Cat: 557235) was purchased from BD Pharmingen (San Diego, CA) Allophycocyaninconjugated anti-mouse CD11b (Cat: 17-0112-82) and the primary rabbit-anti-mouse RAGE (Cat: ab3611-100) were purchased from Abcam (Middlesex, NJ). Secondary goat anti-rabbit-FITC (Cat: sc-2012) and secondary goat anti-mouse-FITC (Cat: sc-2010) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Tumor-associated MPs (CD11b^high CD45^high) and MG (CD11b^high CD45^low) were distinguished based on previously described FACS analysis (21 (link)).

Freshly harvested tumor and peritumoral tissue were minced and digested with trypsin (# 9341 Irvine Scientific, USA) for 20 minutes at 37°C. Tissue homogenate was then filtered through a 40 μm filter and prepped using Fixation/Permeabilization solution according to the manufacturer’s instructions (BD Pharmingen,San Diego, CA). Multiple-color FACS analyses was performed at the City of Hope Analytical Cytometry Core using a 3-laser CyAn immunocytometry system (Dako Cytomation, Fort Collins, CO), and data was analyzed using FlowJo software (TreeStar, San Carlos, CA) as described (27 (link)). PerCP-conjugated antibodies to mouse CD45 (Cat: 557235) was purchased from BD Pharmingen. Allophycocyanin-conjugated anti-mouse CD11b (Cat: 17–0112-82) was purchased from Abcam (Middlesex, NJ). FITC-conjugated anti-mouse MHCII (Cat: 11–5321-82) was purchased from Invitrogen (Carlsbad, CA). Secondary goat anti-rabbit-FITC (Cat: sc-2012) and secondary goat anti-mouse-FITC (Cat: sc-2010) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Tumor-associated macrophages (CD11b^high CD45^high) and microglia (CD11b^high CD45^low) were distinguished based on previously described FACS methodology (28 (link)).