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Murine primary macrophages including peritoneal macrophages and BMDM express similar levels of the BG receptor dectin-1 and exhibit the same ability to respond to Sc BG extracts.

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posted on 2016-02-03, 23:04 authored by Sarah Walachowski, Guillaume Tabouret, Gilles Foucras

(A-B) Freshly isolated 4-days thioglycollate-elicited peritoneal macrophages (TEPM), resident peritoneal macrophages (RPM) and BMDM from WT and Clec7a-/- C57Bl/6, and DBA/2 mice, and the macrophages cell line RAW-Blue, used as control, were assessed for surface expression of dectin-1 by flow cytometry (MACSQuant®, Miltenyi Biotech, Germany). Doublets of cells were excluded at the beginning of the acquisition and a 7-AAD staining was used to discriminate death cells. The surface expression of dectin-1 was determined for each type of primary macrophages on CD115+ cells. (A) Data are expressed as the frequency of dectin-1+ among CD115+ cells. (B) The mean of fluorescence intensity (MFI) of dectin-1 was established on CD115+ cells. Results are presented as the mean ± SD MFI of dectin-1 corrected by either the MFI of isotype control for RAW macrophages, or the MFI of Clec7a-/- C57Bl/6 for WT C57Bl/6 or DBA/2 primary macrophages. (C) Remaining TEPM and BMDM from WT and Clec7a-/- C57Bl/6 mice isolated from these experiments were subcultured and stimulated with 100 μg/mL of crude or enriched Sc BG CW extracts (BG15, BG65 or BG75) or zymosan for 8 h (37°C, 5% CO2). Supernatants were collected immediately after incubation and were assessed for TNFα production by ELISA. Results are shown as the mean ± SD. All data are representative of three mice per group from three independent experiments performed in triplicate. *p < 0.05 (two-tailed unpaired Student’s t-test); mean values not sharing the same letter are significantly different.