2.6. Flow-Cytometry Measurements of Membrane and Intracellular Markers

To identify the membrane markers, anti-human FITC conjugated anti-CD44 and anti-CD105 antibodies were used, both from Miltenyi Biotech (Bergisch Gladbach, Germany). The BJ and A375 cells were plated on 6-well plates (2.5 × 10⁵ cells/3 mL cell culture media), treated for 24 h with 1 and 2 at a subcytotoxic concentration of 50 μM, all samples in duplicates. As reference, untreated cells were growth in the same conditions; instead of Goji extracts, sterile PBS was added to these wells. The cells were harvested, washed with CellWash buffer (from BD Biosciences, San Jose, CA, USA), and 5 × 10⁵ cells were re-suspended in 80 μL PBS with 0.5% FCS. The appropriate amount of antibodies was added, according to the manufacturer’s indication, the cell suspensions were incubated, washed and prepared for flow-cytometry measurement, as described earlier [37].

To evaluate the intracellular protein expression, we used the goat anti-human p65 NF-kβ antibody (acquired from R&D Systems Europe Ltd., Abingdon, UK) with the appropriate PE anti-goat secondary antibody; goat anti-human phosphorilated ERK1+ERK2 (pT202/pY204 and pT185/pY187), phospho p38 (pT180/Y182) and phosphorilated JNK1/2 (pT183/Y185) (all from Abcam, Cambridge, UK). The cells were cultivated and treated as above, and after washing with cold PBS, the cells were permeabilized using the Inside Stain kit from Miltenyi Biotech. Two independent samples were prepared for each treated cell population. The samples were analyzed by flow cytometry, with a FACS Canto II Flow cytometer (from BD Biosciences, San Jose, CA, USA) using the 488-nm, blue, aircooled, 20-mW solid state excitation laser and the 530/30 filter for FITC as well as the 585/42 filter for PE.