Cell staining buffer

Single cells of SVF fraction were resuspended with PBS and stained with zombie NIR™ dye (#423105, Biolegend, USA), blocked with FcX Blocking CD16/32 (#156604), and dyed with a mix of surface marker antibodies. Cells were washed with cell staining buffer (#554657, BD Biosciences, USA), suspended, and transferred to a flow tube. Data was collected on a NovoExpress flow cytometer (Agilent, USA) and analyzed using Flowjo_V10 software (Tree Star).

Swine peripheral blood mononuclear cells (PBMCs) were cultured in 24-well round bottom plates (1 × 10⁶ cells/well) and stimulated for 12 h with PRRSV antigen, recovered by centrifugation and resuspended in 100 µL of PBS containing 10% porcine serum. After washing twice with a cell-staining buffer (BD Biosciences), the cells were suspended in 50 µL of staining buffer and stained with PE-Cy™ 5.5 antipig CD3ε (clone BB23-8E6-8C8, BD Biosciences), APC antipig CD4 (clone 74-12-4, BD Biosciences) and FITC antipig CD8 (clone 76-2-11, BD Biosciences) at 4°C for 30 min. Following another two washes, the cells were suspended in 200 µL of sterile PBS and analyzed using a BD Accuri™ C6 flow cytometer.

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.

Lymph nodes and spleen were separated and grinded to cell suspension. Density gradient centrifugation was used to isolate mononuclear cells. Appropriate concentration of antibodies was added to 100 μl cell suspension at 4 °C for 30 minutes. Cells were then washed twice with PBS and resuspended with cell staining buffer (BD Pharmingen). BD LSRII flow cytometer system (BD Biosciences, USA) was used to test the cell samples. The data was analyzed by Diva and FlowJo 7.6.1.

BMDM, or peritoneal macrophages were collected after treatment with degraders, or after efferocytosis and washed with cell staining buffer (BD Biosciences). Cells were counted and 500,000 cells were collected in 96 well V bottom plates for staining. Macrophages were incubated with Fc block (1:50) (Biolegend) and incubated at 4°C for 15 minutes. Cells were then stained with conjugated antibodies at 1:100 dilution for 30 minutes at 4°C. Macrophages were stained with anti-CD11b-PercpCy5.5 and anti-F4/80-APC (Biolegend) and for MERTK expression, macrophages were also stained with anti-MERTK-PE (Biolegend). Similarly, EO771 and EGFR/TAM cells were collected, washed with cell staining buffer and stained with anti-AXL-PE (Biolegend) or anti-human EGFR-PE antibodies respectively for 30 minutes at 4°C. After staining, all cells were washed 2 times with cell staining buffer and fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature. Cells were washed with flow buffer, transferred into flow tubes and analyzed by BD Fortessa or BD LSR II. Flow cytometry data was analyzed using FlowJo software, version 9.

Single-cell suspensions that obtained from 2D cultured MDA-MB-231 cells or BCSCs were seeded in a confocal dish (for confocal observation) or low-attachment 6-well plates (Corning, Oneonta, NY, USA) at a density of 10,000 cells/well for flow cytometry detection. Cells were cultured overnight before being treated with Doxo (50 nM), AZD5363 (5 μM), and Doxo plus AZD536 for 48 h. After that, the cells were then fixed for 15 min at room temperature with paraformaldehyde (2%) buffer, and permeabilized with Triton X-100 (0.05% in PBS) for 15 min. After triple washing with cell staining buffer (BD, NJ, USA), cells were incubated with anti-FIS antibody (1:50 for flow cytometry detection, and 1:100 for confocal observation) and anti-Mitofusin-1 antibody (1:50 for flow cytometry detection, and 1:100 for confocal observation) primary antibodies for 30 min followed by incubation with anti-mouse APC(1:50), or anti-rabbit PE(1:50) for 30 min before being detected by confocal microscopy, flow cytometry (FACS Calibur, BD), and an ImageStreamX MkII instrument (Amnis, Luminnex), as well as being analyzed with IDEAS Software accordingly.