Rpmi 1640

The cytotoxic activity of the isolated and identified AEG671 as well as the activity of two other samples containing AEGs as the main components was tested. For the purpose the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assays with the application of a human breast adenocarcinoma cell line T47D (Merck KGaA, Darmstadt, Germany) were performed as described by Felczykowska et al. [48 (link)] and Szubert et al. [49 (link)]. T47D cells were plated at 1 × 10⁴ cells per well of 96-well plate containing RPMI1640 (Carl Roth GmbH) medium supplemented with 10% fetal bovine serum (Merck KGaA) and penicillin-streptomycin solution (50 u and 0.05 mg per 1 mL of medium respectively; Merck KGaA) (24 h at 37 °C, 5% CO₂). The cytotoxic effects of tested samples dissolved in 1% DMSO, at final concentrations 25, 50, 100 and 200 µg ml⁻¹ (in culture medium) were examined after 24 h incubation (37 °C, 5% CO₂) using a microplate reader (Spectramax i3, Molecular Devices, LLC. San Jose, CA, USA). Cell viability was calculated as the ratio of the mean absorbance value, for the six replicates containing the samples, to the mean absorbance of the six replicates of the corresponding solvent control, and expressed as a percentage. The results were considered as significant when cell viability decreased below 50%.

Peripheral blood mononuclear cells (PBMCs) were isolated by diluting whole blood with an equal part of RPMI 1640 (Gibco, Thermo Fisher Scientific). Diluted blood was then overlaid onto Ficoll-Paque PLUS (GE Healthcare, Chicago, Illinois, USA) and centrifuged at 450g for 30 min at room temperature. PBMCs were harvested from the interphase using a transfer pipette and added to RPMI 1640. PBMCs were washed twice in RPMI 1640 by centrifuging at 400g for 10 min and discarding the supernatant, following which cell pellets were resuspended in RPMI 1640 containing 10% fetal calf serum (FCS; Gibco, Thermo Fisher Scientific) and enumerated. PBMCs were frozen in freezing media (RPMI 1640+10% DMSO (Carl Roth, Karlsruhe)).

All experiments with human neutrophils were approved by the Ethics Committee of the University Medical Center Göttingen (protocol number: 29/1/17). Neutrophils were isolated from fresh venous blood of healthy donors. Beforehand, all donors were fully informed about possible risks, and the informed consent was obtained in writing; consent could be withdrawn at any time during the study. Blood was received in S-Monovettes EDTA (7.5 mL, Sarstedt, Sarstedt, Germany) and neutrophils isolated according to previously published standard protocols (12 (link),41 (link)). Neutrophils were resuspended in 1 mL HBSS^-Ca2+/Mg2+. Cells were counted and further diluted at the required concentration for the following procedures in RPMI 1640 containing 10 mM HEPES (Roth, Watertown, NY, USA) and 0.5% human serum albumin (Sigma-Aldrich, Burlington, MA, USA). Purity of the isolation was assessed by a cytospin assay (Cytospin 2 Zentrifuge, Shandon, Runcorn, UK) and Diff Quick staining (Medion Diagnostics, West Bengal, India). Cell purity was always greater than 98%.