Evos fl auto 2 fluorescence microscope

TUNEL staining was performed with the In Situ Cell Death Detection Kit, Fluorescein (Roche Applied Science, United Kingdom) according to the manufacturer’s instructions, 6 and 24 h after treatments. Cells were counter-stained with 1 μg/ml Hoechst 33342 (Life technologies). Images of the staining were taken using an EVOS™ fl auto2 fluorescence microscope (Thermo Fisher Scientific) and analysed using ImageJ software to calculate TUNEL positive cells as a percentage of the total population. Flow cytometry measurement of cell death was performed using Annexin V/Draq 7 (Biolegend) co-staining of cells, according to manufacturer’s instructions. Total percentage of double stained cells was used to quantify numbers of apoptotic cells in each condition.

Co-cultures of OECs with EPC-derived CD14-positive monocytes were seeded on fibronectin-coated µ-slide 8 well plates (ibidi, Gräfelfing, Germany) using the same experimental parameters and treated with fucoidan and LPS as previously described (see Section 4.4). After treatment, cells were washed with PBS and fixed using 4% paraformaldehyde for fifteen minutes, followed by three washing cycles of five minutes. For permeabilization, 0.5% Triton^TM-X100 (Sigma Aldrich) was added for fifteen minutes. Cells were incubated with 1% BSA in PBS for thirty minutes to block unspecific binding sites. After three washing cycles of five minutes with PBS, the primary antibody was applied for two hours at room temperature. Human CD68 (MAB20401 R&D Systems) was used as a primary antibody. Antibodies were diluted in 1% BSA/PBS according to the manufacturer’s specifications. Cells were washed three times and then incubated with the secondary antibody Alexa 555 (A31087 Invitrogen) at a concentration of 2 µg/mL for one hour at room temperature. For nuclear staining, 2 µg/mL Hoechst 33528 was applied for eight minutes. After several washing steps, wells were mounted using ibidi Mounting Medium (ibidi) and imaged with the Evos FL Auto 2 fluorescence microscope (Thermo Fisher Scientific).