Tamoxifen tx

To assess the specific role of adipose secretome, the proliferation of mammary cancer cells (MCF-7 and MDA-MB-231) cultured with conditioned media (CM) obtained from the culture of the MA was evaluated using the iCELLigence technology which allows automatic monitoring of cell adherence and proliferation in real-time. CM were collected after 48 h of culture of MA in DMEM/F12 supplemented with FBS (10%) and glutamine (1%). Before use, they were kept under nitrogen atmosphere at −80 °C. After 24 h of adhesion in the iCELLigence system, cells were exposed to CM (dilution 1:1 in fresh complete adipose cell media) and/or tamoxifen (Tx, IC₅₀ = 10 µM, Sigma-Aldrich) for 72 h. The impedance value of each well was measured by the iCELLigence system every 10 min for 72 h and expressed as cell index (CI) values. Data for cell adherence were normalized at 24 h corresponding to the time of treatment to give a normalized cell index. Three independent experiments were conducted.

Tamoxifen (Tx, Sigma-Aldrich) was dissolved in corn oil (Sigma-Aldrich) at a concentration of 20 mg/ml and a single dose of 5 mg/40 gr body weight was administered. When analyzing embryos, Tx was administrated to pregnant females by intraperitoneal injection 24 h post IUE, whereas to analyze adult mice it was administered at perinatal stages.

Quantification of extracellular free DNA was measured by spectrofluorometry. PMNs (1 × 10⁶) resuspended in Hanks' Balanced Salt Solution (HBSS, 136 mM NaCl, 5.3 mM KCl, 5.5 mM glucose, 1 mM MgCl₂, 10 mM HEPES, 1.2 mM CaCl₂, pH 7.4) were incubated in the presence of tamoxifen (TX, 10 μM, Sigma-Aldrich) or DMSO (0.1%, drug vehicle) for 30 min at 37°C with 5% CO₂. Then, to stimulate NETs formation (positive control), cells were incubated with phorbol-12-myristate-13-acetate (PMA; 200 nM, Merck Millipore) for 1 h at 37°C with 5% CO₂. Next, micrococcal nuclease (5U/well, New England Biolabs, Ipswich, MA, USA) was added to each sample, and incubated for 15 min at 37°C with 5% CO₂. Cells were centrifugated at 300 × g for 5 min, and 100 μL of the supernatants were collected and transferred to a 96-well plate. PicoGreen^TM (1:200, Invitrogen, Carlsbad, CA, USA) was added to each well, followed by a 5 min incubation at RT. DNA content and NETs formation was analyzed using an automated Varioskan Flash Reader (Thermo Fisher Scientific, MA, USA) at 485 nm excitation and 530 nm emission.