Monocytes were resuspended at 5 × 106 cells/ml in RPMI 1640 GlutaMAX medium (Gibco, UK) supplemented with 10% FBS (Biochrom, Germany) and 1% Penicillin/Streptomycin (Thermo Fisher Scientific, USA). They were seeded on 6-well plates (VWR International, Germany) and allowed to equilibrate at 37 °C and 5% CO2 for 2 h. Cells were then pre-incubated with 1 μM atRA (Sigma-Aldrich (Germany) or 1α,25 (OH)2D3 (Sigma-Aldrich (Germany) for 30 min. Then, the heat-killed pathogens were added at a pathogen:host ratio of 1:1 for C. albicans yeast and A. fumigatus germ tubes, and 10:1 in case of E. coli stimulation. After 6 h of incubation at 37 °C and 5% CO2, cell viability >90% was assessed by trypan blue staining, and the monocytes were harvested for RNA isolation. The whole experimental workflow is depicted in Supplementary Fig. S1 by Klassert et al.32 (link).
In total, we had four different immune-stimulatory settings (w/o infection, A. fumigatus infection, C. albicans infection and E. coli infection), in each of which we aimed to address the effect of vitamin A (atRA) or vitamin D supplementation.
In total, we had four different immune-stimulatory settings (w/o infection, A. fumigatus infection, C. albicans infection and E. coli infection), in each of which we aimed to address the effect of vitamin A (atRA) or vitamin D supplementation.
Free full text: Click here
