C 12909

Monocytes, isolated from blood as well as commercial monocytes i.e. CD14 + Monocytes from Peripheral blood, single donor (PromoCell, C-12909), were utilized. After thawing, the cells were cultured in a 25 cm² culture bottle in monocyte-to-macrophage medium (Table 1) to differentiate the monocytes towards macrophages. Human serum in the medium differentiates monocytes into macrophages (Andreesen et al. 1983 (link); Musson 1983 (link)). For the first 24 h after thawing, the cells were allowed to recover. Then fresh medium was changed. After 3 days, some of monocytes had attached to the bottom and differentiated to macrophages, whereas some of monocytes were growing in suspension. Every 4–5 days, half of the medium was changed, and the morphology of the cells was monitored by microscope. After 10 days, cells growing were subcultured with ratio of 1:2 into 25 cm² culture bottles. After culturing the cells for 24 days, the macrophages were differentiated and ready to be used in the construction of the in vitro models. Maturity was tested with methods presented in paragraph “Analysis of isolated and differentiated macrophages”. Detachment of macrophages was done by Macrophage detachment solution DXF (PromoCell, C-41330) and scraping with a cell scraper.

Cultivation and siRNA treatment of THP1 cells (DSMZ GmbH, Braunschweig, Germany) was performed as previously reported [7 (link)]. Briefly, the cell line was authenticated via MLL-AF9 breakpoint sequencing, was routinely checked for mycoplasma contamination and transfected with 50 nM Silencer Select siRNAs (Life Technologies, Carlsbad, CA, USA) and Dreamfect (OZ Biosciences, Marseille, France) leading to transfection efficiencies and survival rates of 93 % each. Experimental incubations lasted eight days with repeated transfections on day 0, 3, and 6. Prior to each transfection event, cell densities were determined and cells were reseeded at 5 × 10⁴ cells per ml [7 (link)].
THP1 miRNA mimic transfections were performed as described for siRNA transfections but with 30 nM Ambion Pre-miR miRNA Precursors (hsa-miR-511-5p AM10237, neg Control #1 AM17110, neg Control #2 AM17111) and experimental incubations up to day nine again with repeated transfections on day 0, 3, and 6.
Human CD14+ monocytes (C-12909, PromoCell GmbH, Heidelberg, Germany) were cultivated for 48 h in mononuclear cell medium (PromoCell) prior to immunostaining and RNA isolation. Human CD34+ progenitor cells (C-12921, PromoCell) were directly taken to RNA isolation.