Pe labeled anti cd3e clone 145 2c11

The lungs were digested to single cell suspensions using the Miltenyi Biotec lung dissociation kit as per manufacturer's instructions. Cells were counted to establish the absolute numbers of cells within each lung sample from which subpopulation numbers could be calculated. Cell subpopulations were identified by forward and side scatter profile and subsequently gated using monoclonal antibodies (listed below) along with appropriate isotype controls according to both manufacturer's recommendation and our prior publications (27 (link)).
The fluorochrome-conjugated monoclonal antibodies used here were: PE-labeled anti-CD3e (clone 145-2C11; T-cells), and APC-labeled anti-Gr1 (clone RB6-8C5; Neutrophils) from eBioscience. In additional experiments, neutrophils were also double stained as being Ly6G⁺ CD11b⁺. PD1 and PDL1 antibodies were utilized to co-stain neutrophils for these checkpoint molecules of interest (AbCam). Samples were processed with our lab's Miltenyi Biotec MACSQuant® flow cytometer. FlowJo (version 9.3.2) was utilized to analyze the data.