Easysep mouse pe

The evaluation of GR1⁺ myeloid cells in responsive and non-responsive tumors was performed by single-cell RNA sequencing (scRNA-seq). Briefly, 4T1_P and 4T1_M tumors were prepared as single cell suspensions. Subsequently, GR1⁺ cells were isolated by positive isolation (EasySep Mouse PE, Stemcell Technologies, Cat# 17666). The cells were then washed in PBS with 0.04% BSA and resuspended in 1000 cells/μL PBS. RNA was extracted and immediately was acquired by the 10X Genomics single cell sequencing system, as per manufacture’s instructions. Bioinformatic analysis was then carried out as described below.

GR1⁺ cells were isolated (positive isolation, EasySep Mouse PE, Stemcell Technologies, Cat# 17666) from the spleens of 4T1 tumor bearing mice and cultured overnight with 5% medium containing IFNα and IFNγ (10 ng/ml each, BioLegend Cat# 752802 and Peprotech Cat# 315-05). Subsequently, cells were collected, centrifuged and washed twice with PBS. Ly6E^hi neutrophils were analyzed by flow cytometry and by RT-qPCR as described below. The experimental procedure was carried out as described in the schematic illustration (Figures S4A and S6D) Specifically, Ly6E^hi neutrophils (1x10⁶ cells per mouse) were intravenously injected into mice bearing 50 mm³ 4T1_P or 4T1_M tumors (n=6-7 mice/group), and 4 hours later, mice were treated with anti-PD-1 or IgG control. Ly6E^hi neutrophils were adoptively transferred for a total of 3 times. In some experiments, at the time of the last injection, the cells were first labelled with Live Cell Labeling - Red Fluorescence - (Cytopainter, abcam, Cat# ab187965), in accordance with the manufacture's protocol. Tumor volume was measured twice a week. When tumors reached endpoint, the experiment was terminated.