Ca²⁺ supplementation <i>in vitro</i> increases cell death in polarized human macrophages and favours an M1 phenotype.

Primary human monocyte-derived macrophages were cultured untreated (UT) or polarized into M1 (100 ng/ml LPS, 20 ng/ml IFNγ), M2a (20 ng/ml IL-4), or M2c (20 ng/ml IL-10) macrophages for the indicated hours in either normal RPMI1640 medium (RPMInorm) or RPMI1640 medium supplemented with 1 mM Ca2+ (RPMIsuppl) as indicated. (a-c) The bar graphs show the relative percentages of cells positive for LIVE/DEAD Fixable Blue Dead Cell Stain (LDB+ cells), indicating dead cells, for (a) M1, (b) M2a, and (c) M2c macrophage. (d-f) The expression (mean fluorescent intensity, MFI) of (d) HLA-DRα (UT; M2a, 24 hours) and (e, f) CD86 ((e): UT; M2a, 24 hours; (f): UT; M2c, 24 hours) on live macrophages is shown. The data shown are means ± SEM of biological replicates of six donors, pooled from two independent experiments with similar results. (g-j) The production of (g) TNF and (h) CXCL10 by M1 macrophages (6 hours) and of (i) TGFβ, and (j) IL4 by M2a (48 hours) or M2c (48 hours) macrophages was analysed ICCS. The data shown are means ± SEM of biological replicates of five (TGFβ, IL-4) or six donors (CXCL10, TNF), pooled from two independent experiments with similar results.