Chambered slides

Purified PBMs, seeded at 3 × 10⁶ cells/mL in chambered slides (Ibidi, Germany), were cultured in RPMI supplemented with 10% heat-inactivated FCS, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 100 ng/mL porcine CSF for 3 d. The cells were infected with Benin 97/1 or BeninΔA179L at 0.25 MOI for 16 h. The cells were fixed in 4% paraformaldehyde and washed with PBS, followed by terminal deoxynucleotidyl transferase-dUTP nick end labeling (Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection, Thermo Fisher Scientific), according to the manufacturer’s protocol. The cells were subsequently stained with mouse anti-p30 C18 (63 (link)) for 1 h at room temperature (RT) and the corresponding goat antimouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 488 (Thermo Fisher Scientific), at 1 μg/mL for 1 h at RT. Nuclei were labeled with Hoechst 33342, diluted in PBS at a final concentration of 5 µg/mL, and incubated at RT for 15 min. Imaging was performed using a Leica TCS SP8 confocal microscope with a 40× oil immersion objective. The total number of cells and TUNEL-positive cells was counted using ImageJ 1.x (64 (link)).

Whole culture experiments were conducted using media, PBS, and agonist dilutions in media prewarmed to 37°C in the incubator. Untreated cell supernatant was taken from a culture flask plated and prepared alongside the chambered slides (Ibidi 80826). One hour before the experiment, the media in the wells was changed to DMEM with 10% HIFBS and hoechst stain. At the start of the experiment, cells were treated by diluting the agonist 1:10 in the wells and mixing by pipette and rocking, then incubating at 37°C and 5% CO2 for the indicated stimulation time, diluting the well contents 4 times at 1:6 ratio with warm, untreated media, then incubating further for a total of 30 minutes incubation time. Cells were washed with warm PBS and fixed with Cytofix (BD Biosciences 554655) at 4°C. Fixed cells were washed once more and imaged in PBS. For live-cell imaging, instead of fixing the cells were imaged immediately at 15 minutes.