Anti cd54 apc

Following cell culture, the following molecules were used to stain cells: Live/Dead fluorescent dye (Life Technologies, L23101) to detect cell viability; CellTrace CFSE (Life Technologies, C34554) or Violet (Life Technologies, C34557) to detect cell proliferation; anti-CD25-PE and anti-CD69-PE (BD Pharmingen, San Diego, CA, cat. no. 555432 and 555531 respectively) to detect T cell activation; anti-HLA-DR-PE and anti-CD54-APC (BD Pharmingen, 347401 and 559771 respectively) to measure APC surface markers on HeLa-CIITA cells. To identify cell cycle stages, we fixed T cells in 70% ethanol on ice and used a combined treatment of RNAse incubation and propidium iodide staining (Sigma Aldrich, cat. no. R6148 and P4864 respectively) as previously reported (Vivar et al., 2009 (link)). To measure the amount of phospho-rpS6 ribosomal protein, cells were fixed in Cytofix/Cytoperm solution (BD Biosciences, 554722), permeabilized with Perm/Wash buffer (BD Biosciences, 554723) and stained with an anti-phospho-rpS6(Ser235/236) antibody (Cell Signaling Technology, Danvers, MA, cat. no. 2211L). All experiments were performed with the MACSQuant flow cytometer (Miltenyi Biotech, Bergish Gladbach, Germany).

The HUVECs/edWB coculture was stimulated with 100 ng/ml LPS or an E. coli strain (MOI 50) overnight (Figure 1) or with sDLL1 (5 μg/ml), LPS (100 ng/ml) ± DAPT (1 μM, pretreatment for 1 h), or LPS + anti-DLL1 antibody (5 μg/ml) overnight (Figure 2). After removing edWB and rinsing with phosphate-buffered saline (PBS), HUVECs were analyzed for surface expression of DLL1 (anti-DLL1-APC, Miltenyi Biotec, Bergisch Gladbach, Germany), Notch1 receptor (anti-Notch1-APC, BioLegend GmbH, Koblenz, Germany), ICAM1 (anti-CD54-APC), and E-selectin (anti-CD62E-PE) (both BD Bioscience, Heidelberg, Germany) with antibody staining and flow cytometry. Fluorescence was recorded using the FACS DIVA V 4.12 software on a FACS Canto (BD Biosciences, Heidelberg, Germany).