Dxp athena flow cytometer

The macrophages were washed with DPBS and then incubated with Accutase solution (STEMCELL Technologies) for 15 min at 37 °C. Following incubation, macrophages were detached by robust pipetting, and then the cells were collected and washed with PBS/BSA (PBS + 2% BSA). For surface staining, the cells were directly incubated with antibodies against surface markers. For intracellular staining, the cells were fixed with 4% paraformaldehyde fix solution (Beyotime Biotechnology) and permeabilized with immunostaining permeabilization buffer with Triton X-100 (Beyotime Biotechnology). The cells were then incubated with antibodies against intracellular proteins. At least 5000 cells per sample were analyzed. Samples were acquired with a DxP Athena flow cytometer (Cytek Biosciences, Fremont, CA, USA) and analyzed using FlowJo software (BD Biosciences, Franklin Lakes, NJ, USA). Background signals were controlled by applying a fluorescence minus one (FMO) control.

For cell cycle detection, cells were treated with 10 μM P8 fibrils or P8 fibrils plus 50 nM 17-AAG or STA-9090 for 24 h, followed by treatment with Noc (5 nM) (MedChem Express) for another 24 h. The cells were washed with PBS and fixed with 75% ethanol at 4 °C. The fixed cells were kept at 4 °C for more than 18 h. Before staining, the cells were washed two times with PBS. Then, 200 μL PI/RNase Staining Buffers (BD Biosciences, San Jose, USA) were added to the cells and incubated for 15 min for DNA staining. Then, the cells were detected by flow cytometry. For cell apoptosis detection, cells were cultured and treated with 10 μM P8 fibrils or P8 fibrils plus 50 nM 17-AAG or STA-9090 for 24 h, followed by treatment with ActD (2 μg/mL) (MedChem Express) for another 24 h. Cells were stained with an Annexin V-Alexa Fluor 647/PI kit (#FXP023, 4 A Biotech, Suzhou, China) for apoptosis according to the manufacturer’s instructions. Apoptotic cells were quantified with a DxP Athena™ flow cytometer (Cytek Biosciences, CA, USA) and then analyzed by FlowJo V10 software. Triplicates of all assays were performed.

Apoptosis assays were conducted using the Annexin V-fluorescein isothiocyanate apoptosis kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocols. The results were analyzed with a Dxp Athena™ flow cytometer (Cytek, Fremont, CA, USA).

Spleen was collected from each euthanized mouse 12 hours after surgery and treatment, the single cell suspension was prepared in PBS containing 2% FBS for staining. The cell surfaces were blocked with rat anti-mouse CD16/CD32 antibody for 15 min at 4°C, and then incubated with PE-Cy7-anti-CD3Ɛ, FITC-anti-CD4 and APC-anti-CD25 antibodies (Biolegend, London, United Kingdom) for surface marker staining. After washing, fixation, permeabilization and a second blocking, the intracellular labeling of Foxp3 protein was performed by incubating the cells with PE-anti-Foxp3 antibody for 30 min on ice in the dark. The isotype-matched immunoglobulins (Biolegend, London, United Kingdom) and FMO were used as control for non-specific staining as baseline. The cells were washed twice with fluorescence-activated cell sorting staining buffer and analyzed by DxP Athena™ flow cytometer (CYTEK, USA). All the results were analyzed using FlowJo^R 7.6 software (Treestar, Ashland, OR, USA).

To differentiate the live from the dead cells, cultured BMDM cells were treated with fixable viability dye efluor 510 (BioLegend, USA) for 10 min. After being blocked with Fc receptor blocker (BioLegend, USA) for 10 min, the cells were incubated with FITC-anti-F4/80 (BioLegend, USA), BV605-anti-CD11b (BioLegend, USA) and APC-anti-CD86 (Thermo Fisher Scientific, USA) antibodies for 25 min. To stain CD206, the cells were fixed and permeabilized using a Thermo Fixation/Permeabilization Kit (Thermo Fisher Scientific, USA) and stained with PE-anti-CD206 (BioLegend, USA) for 30 min.
The ratio of apoptotic hypoxic cardiomyocyte cells was determined using Annexin V-AbFluor™/PI (Bestbio, China). Cells were harvested and suspended in binding buffer and then stained with Annexin V for 15 min and PI for 5 min at room temperature in dark conditions. The flow cytometry for these stained cells was performed by a DxP Athena™ flow cytometer (Cytek Biosciences, USA). Data were calculated using FlowJo software v10.5 (FlowJo LLC).

RBL-2H3 cells (ATCC, Manassas, VA) were cultured in MEM (Biological Industries, Israel) with 15% foetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C with 5% CO2 in a humidified atmosphere.
Bone marrow mononuclear cells (BMMCs) were derived from female C57 mice at approximately 4 to 6 weeks of age. Isolated bone marrow progenitor cells were cultured in RPMI 1640 media (Biological Industries, Israel) supplemented with foetal bovine serum (FBS, 10%), penicillin (100 U/mL), streptomycin (100 mg/mL), L-glutamine (2 mM), sodium pyruvate (1 mM), HEPES (10 mM) and recombinant cytokines (stem cell factor, 20 ng/mL; IL-3, 20 ng/mL) at 37°C with 5% CO2 in a humidified atmosphere. After 4 weeks, cultures were composed mainly of MCs (90.1%), as the survival rate and differentiation rate of BMMCs were determined by the Cytek Dxp Athena flow cytometer. Analysis of acquired data was performed with FlowJo software. Analysis of the stained populations was performed by gating on the single (FSCW versus FSC), live cells (FSC versus DYE). Then identify a specific mast cell population expressing both CD117 and the FcϵR (CD117 versus FC) (Figure S1).

An Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme, #A211-01) was used to evaluate apoptosis. The cells were seeded into a six-well plate and cultured in complete medium at 37°C until reaching 80% confluence and then stimulated with 0.05 mM H₂O₂ for 2 h to induce apoptosis. Following digestion with non-EDTA trypsin, cells were collected and stained with PI and FITC for 10 min at room temperature in the dark. A Cell Cycle and Apoptosis Analysis Kit (Beyotime, #C1052) was applied to test the cell cycle. The cells were seeded into a six-well plate and cultured in complete medium at 37°C overnight. After digestion with non-EDTA trypsin, the cells were collected, fixed with 70% ethanol at 4°C overnight and subsequently stained with PI for 30 min at 37°C in the dark. Finally, flow cytometry analysis was carried out on DxP Athena flow cytometer (Cytek Biosciences, USA) equipped with FlowJo Software 6.0 (BD Biosciences, New York, NY, USA).