Acoustic focusing cytometer

PMNs and monocytes were separated as previously described. PMNs and monocytes were incubated with two- fold serial dilutions of recombinant proteins (SpEX or SpEX-M) from concentrations of 50 μg to 1.6 μg in a volume of 500 μl of RPMI medium supplemented with 10% fetal bovine serum in an incubator with 5% CO₂ for 30 min. The supernatant of S. pseudintermedius strain 06–3228 was harvested at log phase to test the toxic effect of secreted, native SpEX. PMNs and monocytes were stained with 1 μl Sytox green (Life Technologies, Inc.) for 30 min, then washed twice with PBS (pH 7.2) and analyzed using a flow cytometer (Attune acoustic focusing cytometer) by gating separately on PMNs and monocytes.
To evaluate the protective effect of canine anti-SpEX-M on PMNs and monocytes, recombinant S. pseudintermedius SpEX, at concentration of 3.1 μg diluted in 1ml of PBS (pH 7.2) was incubated for 30 minutes at 37°C with serum from SpEX-M injected dogs. The cell death cut-off used for flow cytometry analysis was established using leukocytes incubated without SpEX. Mean fluorescent intensity was measured from all gated cells.

Canine blood was collected from healthy dogs using a sterile blood collection system with EDTA anti-coagulant (BD Vacutainer). Then, 600 μl of dog blood was added to 1 ml of red blood cell lysing buffer (Sigma-Aldrich, Cat No. R7757-100ML) for 30 min at 37°C in 15 ml sterile plastic tube, centrifuged and re-suspended in 1ml RPMI medium supplemented with 10% fetal bovine serum. PMNs were incubated with recombinant proteins (LukS-I and LukF-I, LukS-I alone, LukF-I alone, attenuated LukS-I, attenuated LukF-I and 1:2 S. pseudintermedius 06–3228 supernatant) in a volume of 500 μl in RPMI medium supplemented with 10% fetal bovine serum in a 5% CO₂ incubator for 30 min. Supernatant of S. pseudintermedius 06–3228 was harvested at log phase to test the toxic effect of secreted LukS-IR. PMNs were stained with 1 μl of Sytox Green (Life technologies, Inc. Cat No. 1776406) for 30 min, washed with PBS (pH 7.2) twice and analyzed using a flow cytometer (Attune acoustic focusing cytometer). In order to measure the protective effect of anti-LukS-IR on canine PMNs, recombinant LukS-I and LukF-I were incubated with canine anti-attenuated LukS-I LukF-I at a dilution of 1:100 for 30 min at 37°C, then tested with the cell permeability assay as previously described.

Phycoerythrin (PE)-conjugated mouse anti-human CD73, fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD90, PE-conjugated mouse anti-human CD105, and FITC-conjugated mouse anti-human CD34 antibodies were purchased from eBioscience (Waltham, MA, USA). MACS Plex Exosome Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to evaluate the exosome surface markers (CD63 and CD81). PE-conjugated mouse anti-human IDO was treated after permeabilization with 0.1% Triton X-100 in PBS for 10 min. All data were measured with the Acoustic Focusing Cytometer (Thermo Fisher Scientific, Waltham, MA, USA).