Novocyte 2060r flow cytometer

Cells (5 × 10⁵) of the different tumor cell lines were seeded in 6-well plates in a volume of 2 mL, and the following day exposed to the resveratrol isomers and SAHA at the above-mentioned concentrations. As control for apoptosis, STS stock solution (Enzo Life Sciences) was added 20 h before flow cytometry at a final concentration of 5 µM. After 72 h of incubation, cell cycle analysis was determined by PI staining. For this purpose, cells were collected after detachment with trypsin, together with medium and PBS used to wash the plates. Samples were centrifuged at 200 g for 4 min, the supernatant discarded, and the cell pellet washed once with cold PBS (4 °C). Subsequently, the cells were fixated with 1 mL ice cold 75% ethanol and mixed gently, and then placed in the fridge for at least 2 h. Thereafter, 2 mL of cold PBS was added and samples were centrifuged again (200 g, 10 min, 4 °C), followed by carefully aspirating the supernatant. Cells were then washed once with cold PBS and stained with PI/RNAse solution while gently vortexing (PI 50 µg/mL (Sigma–Aldrich) + 100 µg/mL RNAse (Sigma–Aldrich)), and then incubated for at least 20 min at 4 °C in the dark. A NovoCyte 2060R flow cytometer (Agilent Technologies Inc.) was used for measurements; data were analyzed with NovoExpress 1.4.1 software (Agilent Technologies Inc.). For each measurement, 1 × 10⁴ events were counted.

FGF1E-AviTag was labeled with Alexa Fluor 488 C₅ maleimide (Thermo Fisher Scientific) according to manufacturer’s protocol and then biotinylated. U2OS-R1 cells were seeded onto 12-well plates (200,000 cells per well) in full medium and left to attach overnight. Then, medium was removed, cells were washed with PBS buffer and starved with serum-free medium for 4 h. Next, plates were cooled on ice, and labeled FGF1E-AviTag-Biot (500 ng/mL) or labeled FGF1E-AviTag-Biot (500 ng/mL) assembled with non-labeled SA-4A (500 ng/mL) were added to the cells in the presence of heparin, in a serum-free medium supplemented with 1% BSA. After 40 min of incubation on ice, the cells were moved to 37 °C for 15 min to allow for internalization. Then, the medium was removed and the cells were washed with serum-free medium supplemented with 0.2% BSA pH 3.5 (three times, 5 min) and then with PBS buffer (three times, 1 min). Cells were subsequently detached with 10 mM EDTA in PBS buffer, pH 8.0, harvested by centrifugation and resuspended in PBS supplemented with 1% BSA. Cells were analyzed using a NovoCyte 2060R Flow Cytometer and NovoExpress software (ACEA Biosciences, San Diego, CA).

HeLa, MDA-MB-231, SW480 and PC3 cells were seeded in 6-well plates (3.75 × 10⁵ cells per well) and grown overnight. LAF was added to the wells and the cells were cultured for 48 h. Both viable and dead (floating) cells were collected and resuspended using an Annexin V-FITC/PI apoptosis kit according to the manufacturer’s instructions. The analysis was performed using a NovoCyte 2060 R flow cytometer (ACEA Bioscience, Inc., San Diego, CA, USA).

U2OSR1 and SKBR3 cells were treated with 0.5 μg/mL tunicamycin for 24 h or with 50 mM lactose for 15 min before the experiment. The internalization of fluorescently labeled T-Fc (30 nM), Affibody_HER2 (30 nM) and KCK-FGF1.E (30 nM) under various conditions was analyzed according to¹². To investigate the effect of galectins on the internalization of the analyzed proteins, galectin-1 (5 μg/mL) and galectin-3 (5 μg/mL) were added to cells with the tested proteins prior to flow cytometry measurements. Cells were analyzed using a NovoCyte 2060R Flow Cytometer and NovoExpress software (ACEA Biosciences, San Diego, CA, USA).