Anti ly6g pe cy7

Freshly isolated mouse bone marrow cells were cultured in the presence of M-CSF (20 ng/ml) or L929 cell CM for 7 days. Adherent cells were released with trypsin and stained with anti-F4/80-PE (BioLegend, San Diego, CA), anti-CD11b-FITC, anti-CD11c-APC and anti-Ly-6G-PE/Cy7 (BD Biosciences, San Jose, CA). Flow cytometry was performed on a BD LSRII at the UNMC Flow Cytometry Research Facility. Data were analyzed using FlowJo software (FlowJo, Ashland, OR).

Weighed tumors were minced and allowed to digest in a 2 ml mixture of collagenase (400 U type II collagenase, Worthington) and 0.2 mg/ml DNase I in RPMI media at 37°C for 1 hr. The mixture was gently vortexed every 10-20 minutes. The tissue lysate was filtered through a 40 μm mesh prior to immunostaining. The resulting single cell suspension was stained with fixable viability dye eFluor 780, anti-CD45.2 Pacific Blue, anti-CD3 PE-Cy7, anti-CD3 Alexa Fluor 700, anti-Foxp3 Alexa Fluor 700, anti-CD11c eFluor 615, anti-NK1.1 PE (all from eBioscience), anti-Granzyme B APC and anti-CD4 Qdot 605 (from Life Technologies), anti-CD8 Brilliant Violet 650, anti-CD11b Brilliant Violet 570, anti-CD19 Brilliant Violet 650, anti-F4/80 FITC (all from BioLegend), anti-Ly6C APC, anti-Ly6G PE-Cy7, and anti-Ki67 PE (BD Biosciences). The percent positive cells were analyzed by FlowJo and gated on CD45 positivity. To analyze the number of CD133⁺CD44⁺ cells, the single cell suspension was incubated in the dark, on ice with Aqua LIVE/DEAD Fixable Dead Cell Stain (Molecular Probes) 1:1000 for 30 min, followed by staining with anti-CD44 APC (eBioscience, 1: 400) and anti-CD133 PE (eBioscience, 1: 200). Unstained, LIVE/DEAD only, and single stain served as control. Doublets were gated out using forward scatter width/height and sideward scatter width/height event characteristics.