Amicon ultra 10 k centrifugal filter units

The cytokine levels were measured by ELISA in media conditioned by spheroids or by complete skin models. Secretion of angiopoietin-2 (ANG-2) was measured in conditioned media incubated 48 hours with the complete melanoma model using a DuoSet ELISA kit (R&D Systems, Minneapolis, MN). Quantification in spheroid supernatants (48 h) was performed during their culture in GravityTRAP^TM plates (InSphero AG, Schlieren, Switzerland) using VEGF-A and VEGF-C DuoSet ELISA kits (R&D Systems). Supernatants were frozen and kept at −80 °C. CCL21 quantification was performed using a DuoSet ELISA kit (R&D systems) on a 48 h supernatant previously concentrated using 0.5 mL Amicon Ultra 10 K centrifugal filter units (Millipore, Billerica, MA, USA). Medium incubated without cells was used as a negative control.

Serum samples were obtained from four healthy adult volunteers—students from the Faculty of Pharmacy, Carol Davila University of Medicine and Pharmacy. Ethical approval for collecting the peripheral venous blood from human subjects was obtained from the Carol Davila University of Medicine and Pharmacy Ethics Committee. Serum lipoprotein concentrates were isolated from each serum sample following centrifugation at 7000 x g, 4°C, 60 min, on Amicon Ultra-10 k Centrifugal Filter Units 10.000 NMWL (Millipore). The assessment of lipid hydroperoxides in serum lipoprotein concentrates using the Amplex Red fluorescent probe was performed according to the method previously described [52 (link), 53 (link), 55 (link)].
Briefly, 50 μL of serum concentrate samples (standardized for a protein content of 0.5 mg protein/mL phosphate buffer 20 mM, pH 7.3) was incubated for 10 minutes at room temperature (25°C) with different procaine and GH3 concentrations: 0 (control), 0.5, 1.0, 2.0, 5.0, and 10 mM. After the addition of 50 μL Amplex Red working solution (300 μM), the samples were incubated for 10 minutes at room temperature and protected from light. The samples were diluted to 2000 μL with PBS 1x before the fluorescence measurements as above described. Results were also expressed as lipid peroxidation inhibition in percentage (%).