Fluorescent Labeling of Monocyte Adhesion

Following density gradient separation, peripheral blood mononuclear cells from a healthy donor were harvested and monocytes were isolated using human CD14 MicroBeads UltraPure (130-118-906; Miltenyi Biotec, Germany), according to the manufacturer’s instructions.^{25 (link)} CellTracker Red CMTPX (C34552; Invitrogen) at a concentration of 2 µM was used to fluorescently label the monocytes, according to the manufacturer’s instructions. ECs were cultured under flow using the orbital shaker model as described above and stimulated with TNF (10 ng/mL) for the last 4 hours of flow. The media was removed and replaced with media containing fluorescently labeled monocytes (1×10⁶ per well) and the cells were incubated for 2 hours under static conditions. The wells were washed gently with PBS to remove nonadherent monocytes and fixed in paraformaldehyde (4% w/v). DAPI (Sigma) was used to label the nuclei. Fluorescent images were taken using the ×20 objective of a wide-field microscope (DM14000B; Leica) to detect adherent monocytes. An average of 6 images were analyzed per well and used to calculate the average number of adherent monocytes per EC per sample.