Facsverse flow cytometer

To detect the expression of CD46, CD55, or CD59, flow cytometry analysis was performed, as described previously^{15 (link)}. The collected cells were reacted with FITC-conjugated anti-CD46, anti-CD55 or anti-CD59 Abs (Medical & Biological Laboratories) for 1 h at 4 °C. Stained cells were washed twice and analyzed using a FACSVerse flow cytometer (BD Biosciences). Data acquisition was performed on a FACSVerse flow cytometer (BD Biosciences) with BD FACSuite software.

Transfection efficiencies and cell viabilities were determined in COS-7 cells. Cells were transfected with mCherry and SuperNova constructs in a 6-well plate (2.5 × 10⁵ cells/well), trypsinized 24 h post-transfection, washed and resuspended in 100 µL PBS for flow cytometry analysis with a BD FACSVerse flow cytometer (BD Biosciences). Untransfected cells were used to apply gating and to discriminate between SuperNova-positive and negative cells. For cell viability evaluation, the culture medium of the cells was changed to phenol-red free prior to illumination of the SuperNova. SuperNova was activated with 590 nm light for 24 h. Cells were collected in 96-wells v-bottom plates by trypsinization after treatment and stained with Zombie Violet fixable viability dye (BioLegend, San Diego, CA, USA; cat# 423113; 1:2000 dilution v/v). Cells were fixed in 4% PFA for 5 min, washed with PBS containing 1% BSA and 0.05% sodium azide (PBA) and resuspended in 100 µL PBA for flow cytometry analysis with a BD FACSVerse flow cytometer (BD Biosciences). Flow cytometry data was analyzed using FlowJo X (FlowJo LLC, Ashland, OR, USA).

The isolated immunocytes were blocked with purified rat anti-mouse CD16/32 antibodies (BD Biosciences, USA) for 30 min at 4°C, then stained with antibodies for 30 min at 4°C, washed and analyzed by a BD FACSVerse flow cytometer (BD Biosciences, USA). The following monoclonal anti-mouse antibodies were used: anti-CD11b-BV421, anti-LY6C-APC, anti-LY6G-PE, anti-CD3-PerCP, anti-CD4-APC-H7, and anti-CD8-FITC (BD Biosciences, USA). The labeling cells were assessed using a BD FACSVerse flow cytometer (BD Biosciences, USA). The acquired data were analyzed using FlowJo 7.6 software (TreeStar, Inc.).

For T cell proliferation analysis, PBMCs from Tg and WT pigs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) dye (BioLegend) as previously described (44 (link)). Briefly, cells were incubated with 5 µM CFSE in PBS containing 5% FBS at 37°C for 5 min and washed three times with ice-cold PBS-5% FBS. 2 × 10⁵ CFSE-labeled PBMCs/well of a 96-well plate were cultured in RPMI-1640 media supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin in the presence of stimulation. PBMCs were harvested after a 3-day culture for subsequent analysis by the BD FACSVerse™ flow cytometer.
Peripheral blood mononuclear cells were cultured in 24-well round bottom plates (1 × 10⁶ cells/well) and stimulated for 0, 24, 48, and 72 h with ConA (2 µg/mL), recovered by centrifugation and re-suspended in 100 µL of PBS containing 10% porcine serum. After being washed twice with a cell-staining buffer (BD Biosciences), the cells were suspended in 50 µL of staining buffer and stained with mouse anti-pig 4-1BB monoclonal antibody at 4°C for 30 min. After two washes, they were stained with FITC anti-mouse IgG (BioLegend Cat. No. 406001). Then, the cells were washed two times, suspended in 200 µL of sterile PBS, and analyzed using the BD FACSVerse™ flow cytometer.