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Abstract This study explores methods to isolate high-pure monocytes and optimize the best growth factor concentration to generate monocytes-derived dendritic cells (mo-DCs), subset DC1, which is crucial in immune responses. Three protocols for monocyte isolation from peripheral blood mononuclear cells (PBMCs) were evaluated: three-hour incubation on FBS-coated flasks; an overnight incubation on FBS-coated flasks; and Magnetic Activated Cell Sorting (MACS). Additionally, five different concentrations of human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) and human recombinant interleukin-4 (hrIL-4) were compared. We used Flow cytometry to assess the isolation, purification, and generation of pure monocytes characterized as CD14+, and expression of mo-DC classical markers (HLA-DR, CD80, CD83, and CD86). The obtained results show that monocytes isolated with the second method (overnight incubation) had the highest purity (P 0.05), balancing purity and cost-effectiveness. A combination of hrGM-CSF and hrIL-4 at 400 U/mL produced the most favorable outcomes, leading to the highest rate of mo-DC generation (P

Abstract Recognizing new anticancer compounds to improve Breast cancer treatment seems crucial. Essential oil of Zataria Multiflora (ZEO) is a secondary metabolite with some biological properties, yet underlying cellular and molecular anticancer properties of ZEO is unclear. GC/MS analysis revealed that carvacrol is the major ingredient of the essential oil. ZEO increasingly suppressed viability in MDA-MB-231, MCF-7 and T47D Breast cancer cells while nontoxic to L929 normal cells in monolayer cell cultures (2D), whereas MDA-MB-231 multicellular spheroids (3D) were more resistant to inhibition. ZEO significantly induced cell apoptosis confirmed by fluorescent staining, flow cytometry analysis and DNA fragmentation in MDA-MB-231 2D and 3D cell cultures. ZEO increased ROS generation and subsequent loss of ΔΨm, caspase 3 activation and DNA damage which consequently caused G1 and G2/M cell cycle arrest in a dose- and time-dependent manner in 2D. S phase arrest occurred in cell spheroids therefore ZEO possible DNA interaction with gDNA was investigated and revealed ZEO binds DNA via intercalation. Altogether, these data corroborate anticancer properties of ZEO and suggest that cell culture format (2D monolayer vs. 3D spheroid) plays a critical role in drug response and provides new insights into the mechanisms underlying ZEO cytotoxicity effect on Breast cancer cells.