Canine fc receptor binding inhibitor

Passage 2 AT-MSCs were analyzed by flow cytometry. The cells were placed in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences; 2 × 10⁵ cells/tube), washed with FACS buffer (PBS containing 2% FBS), blocking Fc receptors with canine Fc receptor binding inhibitor (Thermo Fisher Scientific), and then incubated with the following fluorescein- (FITC-) or phycoerythrin- (PE-) conjugated antibodies: anti-CD14-FITC (BD PharMingen), anti-CD29-PE (BioLegend), anti-CD34-PE (R&D Systems), anti-CD44-PE (BioLegend), anti-CD45-FITC (eBioscience), and anti-CD90-PE (eBioscience) or their respective isotype controls listed in Table 1. The cells were washed twice with FACS buffer and resuspended in 500 μl FACS buffer. Fluorescence was evaluated by flow cytometry in a FACSCalibur instrument (BD Biosciences). Data were analyzed using WinMDI 2.9 analysis software.

PBMCs were prelabeled with a 5 μM carboxyfluorescein succinimidyl ester (CFSE) solution using a CFSE Cell Division Tracer Kit (BioLegend, Tokyo, Japan) before seeding and stimulation with ConA. PBMCs were cultured with naive or primed EVs at various concentrations (1, 5, and 10 μg/ml). After 4 days, PBMCs were collected and washed with FACS buffer (PBS with 2% FBS). To inhibit nonspecific binding, canine Fc receptor binding inhibitor (Thermo Fisher Scientific) was added to cells, followed by incubation on ice for 20 min. After blocking, PBMCs were stained with anti-CD4-APC (clone: YKIX302.9, eBioscience, Tokyo, Japan) or the isotype control. The proliferation of PBMCs and CD4⁺ T cells among PBMCs was measured by flow cytometry.

5 × 105–106 cells were stained in round bottom 96 well plates. Surface antibodies were diluted with staining buffer (2% FBS, 1 mM EDTA, and 0.02% NaN3 in PBS) and blocking buffer using canine Fc receptor binding inhibitor (ThermoFisher, #14–9162–42) and canine gamma globulin (Jackson ImmunoResearch, #004–000–002). Details regarding specific antibodies utilized in this study can be found in S2 Table. For intracellular staining of canine interferon-γ and granzyme B, cells were mixed with viability stain, then washed and incubated with fixation and permeabilization solution per manufacturer’s instructions (BD Biosciences). We then incubated with intracellular stain or isotype prepared in Perm/Wash Buffer^™ followed by centrifugation and resuspension in 1% paraformaldehyde for flow cytometry analysis. Fluorescent minus one (FMO) controls containing all the fluorochromes in the panel except the one for that marker of interest were used to control for fluorescent spread due to multiple fluorochromes in a given panel.

The cell surface markers were analyzed with flow cytometry. The primary ADSCs at passage 3 (n = 3), and the ADSC-K4DT (n = 3) and ADSC-K4D (n = 3) cells were analyzed at low (approximately PDL 20), intermediate (approximately PDL 50), and high (approximately PDL 100) PDs. The cells were washed with FACS buffer (PBS containing 2% FBS), and the Fc receptors were blocked with canine Fc receptor binding inhibitor (Thermo Fisher Scientific). Then, the cells were incubated with the following phycoerythrin (PE)-conjugated antibodies: anti-CD29-PE (clone: TS2/16; BioLegend, San Diego, CA, USA), anti-CD34-PE (clone: 1H6; R&D Systems, Minneapolis, MN, USA), anti-CD44-PE (clone: IM7; BioLegend), anti-CD45-PE (clone: YKIX716.13; eBioscience, San Diego, CA, USA), anti-CD90-PE (clone: YKIX337.217: eBioscience), and anti-HLA-DR-PE (clone: G46-6: BD Biosciences, Franklin Lake, NJ, USA). All the cells at each passage were examined in triplicate.