Cd11b clone m1 70

Single-cell suspensions from muscle were prepared by collagenase II (Worthington Biochemical Corp, Lakewood, NJ, USA) digestion followed by staining with fluorochrome-conjugated antibodies.
Antibodies used: (1) Gr1 Clone RB6-8C5 (eBioscience, ThermoFisher, Waltham, MA, USA, Cat#48-5931-82); (2) F4/80 Clone BM8 (Invitrogen, Cat# 11-4801-82); (3) CD11b Clone M1/70 (Invitrogen, ThermoFisher, Waltham, MA, USA, Cat#17-0112-81); (4) Siglec-F Clone E50-2440 (RUO), (BD BioSciences, ThermoFisher, Waltham, MA, USA, Cat# 552126); (5) CD206 Clone C068C2 (BioLegend, San Diego, CA, USA, Cat#141701) and live dead stain (eBioscience). Data acquisition was performed on a FACS Aria or Fortessa (BD Biosciences). UltraComp eBeads (Cat#01333342, Invitrogen) were used to generate single-stain controls. Data was analyzed using FlowJo software (version 10.2) and gating strategies as previously described [39 (link)] to discriminate against dead cells, debris, and doublets were utilized. M1 macrophages were defined as Gr1^low-med, F4/80⁺, and CD11b⁺.

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.