2.2. Experimental Design

In all experiments on shear flow on monocytes’ functions from different donors (n = 20, in triplicate), cell repellent plates (Greiner Bio-One, Leipzig, Germany) were used to avoid unwanted adhesion to the plastic surface. The experimental design is illustrated in Figure 1A. After isolation, the cell number and viability were measured using the CASY cell counter (Omni Life Science, Bremen, Germany). Afterward, the monocytes were diluted in cell culture medium and incubated as a suspension culture under either static or dynamic conditions on an orbital shaker (Celltron, Infors AG, Bottmingen, Switzerland) with 150 rpm according to previously described experimental models for shear flow [12]. After an overnight resting period, monocytes were stimulated with GM-CSF for 24 h (10 ng/mL, PAN-Biotech, Aidenbach, Germany) [6] or left unstimulated using triplicates for each treatment. According to described procedures (6, 7), ex vivo culture was stopped by transferring the multiwell plates to 4 °C for 30 min. After that, non-adherent monocytes were harvested by centrifugation (300× g for 5 min). The cells were counted with the CASY cell counter, followed by dilution to necessary concentrations before the intended evaluation of adherence, migratory capacity, and metabolic activity. Supernatants were collected and stored at −20 °C until the measurement of cytokines. In addition to shear flow-related effects, potential impairment of functionality related to the duration of experimental procedures was evaluated. Thereby, cell size adherence, migratory ability, and metabolic rate were examined directly after isolation and compared to the results observed after overnight resting and 24 h culturing in cell culture medium.

Evaluation of shear flow-mediated effects on non-adherent classical monocytes. (A) After isolation, CD14⁺/CD16^- monocytes were cultured under dynamic (=shear flow) or static conditions with or without (w/o) GM-CSF stimulation. Cytokine release was measured in cell supernatants after culture. After harvesting, naïve and GM-CSF-activated monocytes were applied to functional tests regarding their migratory capacity, adherence, and metabolic activity. (B) The number, size, and viability of isolated monocytes were measured directly after isolation and after culture using the CASY cell counter & analyzer. A representative result of the gated monocyte population is shown based on cell diameter, cell counts, and viability.