Rea control s pe

After lipid exchange as described above, cells were dislodged from plates using enzyme-free cell dissociation solution (Millipore), spun down at 300g, 4 °C for 5 min, and washed with blocking buffer (0.5% BSA, 2 mM EDTA in PBS) at room temperature. Manufacturer’s protocol was followed for fluorescent antibody staining. Cells were resuspended in 100 μl blocking buffer, CD220-PE antibodies against IR alpha chain or recombinant antibody (REA) control (S)-PE (Miltenyi Biotech) were added, and the samples were incubated at 4 °C in the dark for 10 min. Samples were washed at room temperature in PBS blocking buffer again. Flow cytometry analysis was performed using FACSCalibur (BD Bioscience) collecting 10,000 events per sample. Data was analyzed using FlowJo version 10 software.

CB-ECFCs were pre-treated for 24 h with 0.01, 0.1, 1, 10, 50 and 100 ng/ml of TNFα. Untreated CB-ECFCs from the same donors and passages were used as control group. Then, cells were detached using cell dissociation reagent TrypLE (Gibco) and proceeded to immunostaining using mixes of following antibodies: FITC-anti-CD31, VioBlue-anti-CD144, PE-Vio770-anti-VEGFR2 (KDR), APC-anti-TNFR1, PE-anti-TNFR2, APC-anti-ICAM (CD54), PE-anti-VCAM (CD106), APC-anti-TIE2 (CD202b), REA control (s)-APC, REA control (s)-PE (Miltenyi-Biotec). Events acquired on a LSRFORTESSA flow cytometer (BD-Biosciences) and analyzed using FlowJo software v10 (FlowJo-LLC).

Cells were harvested after differentiation and washed thrice in autoMACS running buffer (Miltenyi Biotec, #130-091-221, Gladbach, Germany). Cells were transferred to 96-well plates with 2 × 10⁵ cells for each antibody panel. Cells were stained with the following antibodies: diluted 1:50, REA Control (S)-VioGreen (Miltenyi Biotec, #130-113-444), REA Control (S)-PE (Miltenyi Biotec, #130-113-438), REA Control (S)-APC (Miltenyi Biotec, #130-113-434); REA Control (S)-PE-Vio770, (Miltenyi Biotec, #130-113-440); HLA-DR-VioGreen (Miltenyi Biotec, #130-111-795), CD54-APC (Miltenyi Biotec, #130-121-342); CXCR4-PE-Vio770 (Miltenyi Biotec, #130-116-161); CD11b-VioGreen (Miltenyi Biotec, #130-110-617); CD83-PE (Miltenyi Biotec, #130-110-561); CD86-APC (Miltenyi Biotec, #130-116-161) for 10 min at 4 °C in the dark. Afterwards, cells were washed thrice with autoMACS running buffer and stained with DAPI (Sigma, #D9542), to exclude dead cells for the determination of the cell viability.