Characterization of Middle Ear Immune Cells

Monoclonal antibodies, labeled with appropriate fluorochromes were purchased from BD Biosciences (San Jose, CA, USA), Invitrogen (Carlsbad, CA, USA), or Life Technologies (Carlsbad, CA USA). Cells isolated from the middle ear effusion were stained for Ly6G (clone 1A8), Ly6C (clone AL-21), NK1.1 (clone PK136), CD45R (B220 clone RA3-6B2), CD11c (clone HL3) (BD Biosciences, San Jose, CA, USA), CD11b (clone M1/70) (Invitrogen, Carlsbad, CA, USA), and live/dead markers, including nucleic acid Sytox dyes and amine-reactive dyes (Life Technologies, Carlsbad, CA USA). This allowed discrimination of neutrophil subsets based on staining of Ly6G: CD11b+ CD45R− NK1.1− Ly6G^hi mature cells; CD11b+ CD45R− NK1.1− Ly6G^int immature cells. Macrophages were identified as CD11b± CD45R− NK1.1− Ly6G− Ly6C+; and dendritic cells as CD11c+^{48 (link)}. Cells undergoing phagocytosis were identified as positive for surface markers of subsets as above and also positive for the bacterial reporter GFP. Cells were analyzed on an LSRII flow cytometer (BD Biosciences, San Jose, CA, USA) and analysis was performed using FlowJo (BD Biosciences, San Jose, CA, USA). All samples were analyzed the same day as the samples were collected and cell identification by sequential gating performed on live cells with doublet discrimination.