Human umbilical vein endothelial cells (HUVECs, CRL-1730) were purchased directly from ATCC, seeded at 4x105 cells/well and cultured for 18 hours to adhere in 24-well plates. After adherence, HUVEC media was removed and replaced with CM from Effectene (CM) or Effectene+cffDNA-stimulated monocytes or PBMCs (“cffDNA CM”). For each well of HUVECs, CM from 2 wells of stimulated monocytes or PBMCs was used (2:1 ratio). For select HUVEC wells, neutralizing antibodies for IL-1β (clone: 2805R, 5 µg/mL, R&D Systems) or TNF-α (clone: 28401, 10 µg/mL, R&D Systems) were added to monocyte or PBMC CM, respectively. HUVECs were incubated for 18 hours in monocyte or PBMC CM.
After incubation with monocyte or PBMC CM, HUVECs were harvested for staining with fluorescent antibodies for E-selectin (clone: HCD62E, BioLegend, San Diego, CA) and ICAM-1 (clone: HA58, BioLegend). Cells were stained in 100 uL of FACS buffer using 2 uL of each antibody per reaction for 30 minutes at 4°C. After staining, cells were washed and resuspended in 50 uL of PBS and imaged using the ImageStream X MKII (Luminex) which provides confocal images and flow cytometry analysis. Events were gated on single cells as determined by size and appearance. 10,000 single cells were collected from each sample per experiment for analysis using ImageStream IDEAS software (Luminex).
After incubation with monocyte or PBMC CM, HUVECs were harvested for staining with fluorescent antibodies for E-selectin (clone: HCD62E, BioLegend, San Diego, CA) and ICAM-1 (clone: HA58, BioLegend). Cells were stained in 100 uL of FACS buffer using 2 uL of each antibody per reaction for 30 minutes at 4°C. After staining, cells were washed and resuspended in 50 uL of PBS and imaged using the ImageStream X MKII (Luminex) which provides confocal images and flow cytometry analysis. Events were gated on single cells as determined by size and appearance. 10,000 single cells were collected from each sample per experiment for analysis using ImageStream IDEAS software (Luminex).