Pe anti human cd54

The differentiation of monocytes to macrophages and polarization towards M1 and M2 was performed as previously described (Werz et al., 2018 (link)). M1 were generated by incubating monocytes with 20 ng/ml GM-CSF (Peprotech, Hamburg, Germany) for 6 days in RPMI 1640 supplemented with 10% FCS, 2 mmol/L L-glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml), followed by 100 ng/ml LPS and 20 ng/ml INF-γ (Peprotech) treatment for another 48 h. M2 were obtained by incubating monocytes with 20 ng/ml M-CSF (Peprotech) for 6 days and subsequent treatment with 20 ng/ml IL-4 (Peprotech) for additional 48 h. Correct polarization and purity of macrophages were routinely checked by flow cytometry (FACS Canto Plus flow cytometer, BD Bioscience) as previously reported (Werner et al., 2019 (link)) using the following antibodies: FITC anti-human CD14 (2 µg/test, clone M5E2, BD Bioscience), PE anti-human CD54 (1 µg/test, clone HA58, BD Bioscience), APC-H7 anti-human CD80 (0.25 µg/test, clone L307.4, BD Bioscience), PE-Cy7 anti-human CD163 (2 µg/test, clone RM3/1, Biolegend, San Diego, CA), and PerCP-eFluor710 anti-human CD206 (0.06 µg/test, clone 19.2, Biosciences, San Diego, CA).

To analyze the cell cycle and measure CD25 expression, peripheral T cells (10⁶/ml) were stimulated with for 72 h anti-CD3 and anti-CD28 antibodies in 24 well plates in a total volume of 2 ml complete medium in the presence or absence of different concentrations of VCE-003. The expression of CD25 and CD54 at the cell surface was measured by fluorescence using a specific mAb (CD54: PE anti-human CD54; BD Pharmingen; San Diego, CA, USA; CD25: Mouse Anti-Human interleukin-2 receptor; Dako; Glostrup, Denmark), which was analyzed by flow cytometry in a FACSCAnto II flow cytometer. To analyze the DNA profile, the cells were washed in PBS and fixed in ethanol (70%, for 24 h at 4°C), before digesting their RNA (RNAseA, 50 U/ml) and staining them with propidium iodide (20 µg/ml) to be analyzed by cytofluorimetry. Ten thousand gated events were collected per sample and the percentage of cells in each phase of the cell cycle was determined. To analyze cell division, purified T cells were stained with carboxy-fluorescein succinimidyl ester (CSFE) and stimulated with anti-CD3 (coated 1 µg/ml) and anti-CD28 (coated 0.5 µg/ml) antibodies in the presence or absence of VCE-003 for 6 days and the percentage of proliferating cells (defined as weak CFSE fluorescence) was determined by flow cytometry.