Neutrophils are rapidly recruited to the dermis where they phagocytose BCG and then massively crawl out from the skin. (A) Cells crawling out from ear skin explants from 4 to 72 hours after BCG-egfp vaccination were analyzed by flow cytometry after CD11b staining. (B) Twenty-four hours after vaccination, EGFP⁺ skin explant cells gated on a side-scatter (SSC-H), EGFP dot plot were phenotyped as CD11b⁺ Ly-6G⁺, MHCII^-, and F4/80^-. (C) CD11b⁺/EGFP⁺ cells were sorted by flow cytometry and observed by light microscopy after staining with May-Grünwald-Giemsa. Characteristic polylobed nuclei confirmed that main BCG host cells in skin are neutrophils. In most cases, several bacilli were detected inside each neutrophil (arrows). Cytocentrifuged cells were observed under a Zeiss Axioskop light microscope with an Achroplan 100 ×/1.25 NA oil iris objective. Images were acquired with a Leica 300F camera and processed with Leica Qwin software. (D i-ii, iv-vi) Ear skin cryosections were immunolabeled with anti-Ly-6G (red). Neutrophils that invaded the dermis from 4 to 72 hours after injection were focally organized around bacilli at the injection site. Images were acquired as in Figure 3B. (iii) At 12 hours, bacillus (green) and neutrophil (red) colocalization was observed by confocal microscopy. After labeling with Alexa 594 slides were analyzed under a Zeiss Axioskop 2FS microscope with a Plan-APOCHROMAT 63 ×/1.4 NA objective. Images were acquired and processed with Zeiss LSM 510 software.