Figure 1.

Gas6/ProS-TAM system expression in mouse macrophages. (a) Identification of mouse primary peritoneal macrophages. These cells were identified by immunofluorescence staining for F4/80 using anti-F4/80 antibodies and counter-stained with DAPI for the nuclei (left panel). Negative controls were incubated with pre-immune rabbit serum instead of the anti-F4/80 antibodies and counterstained with DAPI (right panel). (b) The purity and viability of macrophages were quantitatively assessed by flow cytometry after double staining with phycoerythrin (PE) -conjugated antibodies against F4/80 (F4/80 Ab-PE) and fluorescein isothiocyanate (FITC) -conjugated annexin V (AnxV-FITC). The living macrophages were determined to be an F4/80⁺/AnxV^– population (left panel). An isotype control was obtained by labelling cells with FITC-conjugated rat IgG2a (FITC-IgG2a) (right panel). (c) Expression of Gas6, ProS and TAM receptors in the macrophages. Total RNA was extracted from the macrophages and the relative mRNA level was analysed using quantitative PCR. (d) Western blotting for detection of TAM receptors. β-Actin was used as the loading control. (e) Gas6 and ProS production by the macrophages. The macrophages were cultured in a serum-free medium for 24 hr. Gas6 and ProS concentration in the medium was measured by ELISA. These images are representatives of at least three independent experiments. Data represent the mean ± SEM of three experimental values (n = 1 mouse per experiment). *P < 0·05; **P < 0·01.