Cd107a alexafluor647

The same two lobes of the right lung were harvested from each A/J mouse (n = 6/group) for flow cytometry and incubated in digestion media consisting of collagenase (300 U/ml, Sigma), dispase (1 U/ml, Worthington), and DNAse (2 U/ml, Calbiochem) for 30 min at 37 °C with stirring. Cells were then passed through a 40 µm cell strainer (BD Falcon), and red blood cells eliminated with lysis solution. Single cells were resuspended in a solution of Brilliant buffer (BD Bioscience) and stained for 30 min at 4 °C with the following antibodies: CD45-Brilliant violet 510 (30F11), CD24-Brilliant violet 604 (M1/69), CD64-Brilliant violet 711 (X54-5/7.1), AI/AE- Brilliant violet 650 (M5/114.15.2), CD8-Alexafluor 700 (53–6.7), CD4-FITC (GK1.5), LyC6/LyG6-APC (rb6-8C5), CD11b-PE-Cy7 (M1/70), CD11c-PE-Cy5 (N418), CD206- Brilliant violet 421 (C068C2), CD69- Brilliant violet 785 (41.2F3), CD25-PE (3C7), CD107a-Alexafluor 647 (1D4B), and 5 μg/ml anti-mouse CD16/CD32 antibody (Biolegend) to reduce antibody binding to Fc receptors. Zombie NIR staining was used to exclude dead cells. Cells were analyzed using a Cytek Aurora equipped with 5 lasers (UV 355 nm, violet 405 nm, blue 488 nm, yellow-green 561 nm, and red 640 nm) and FlowJo x.10.0.7r2 software (Tree Star). The gating strategy used and shown in Supplemental Fig. 6 was adapted from a published protocol^{78 (link)}.