Cd117 apc

Stomach intraepithelial leucocytes were prepared as described above. Cells were incubated with ZombieFITC (1:500, BioLegend) for dead/live discrimination and 10 μg ml⁻¹ anti-CD16/32 antibodies (39, BioLegend) to block Fcγ receptors for 15 min at room temperature. After washing with PBS supplemented with 5% FCS (Sigma-Aldrich), cells were stained with CD45 BV785, CD117 APC and IgE BV421 (R35-72, BD Bioscience) for 20 min on ice and protected from light. After washing and centrifugation, cells were fixed and permeabilized using a FoxP3-intracellular staining kit (BioLegend) according to the manufacturer’s instructions. Cells were stained with 0.11 μg ml⁻¹ anti-5-HT (5HT-H209, Dako) or isotype control antibodies for 30 min, washed in PBS and stained with anti-mouse-IgG1 (RMG1-1, BioLegend) antibodies for 30 min before analysis with a BD LSRFortessa (Becton Dickinson).

The following antibodies were used in flow cytometry: B220 FITC 1:50 (BD Pharmingen, RA3-6B2), CD3 BV421 1:200 (17A2, BioLegend), CD3 FITC 1:50 (17A2, BD Pharmingen), CD3 PE-Cy-7 1:25 (145-2C11, BD Pharmingen), CD11b PerCP-Cy5.5 1:400 (M1/70, eBioscience), CD11b BV421 1:400 (M1/70, BioLegend), CD11b PE-Cy-7 1:400 (M1/70, eBioscience), CD11c BV421 1:100 (N418, BioLegend), CD16/32 unconjugated 10 μg ml⁻¹ (93, BioLegend), CD19 BV421 (6D5, BioLegend), CD19 APC 1:400 (1D3, BD Pharmingen), CD45 BV421 1:400 (30-F11, BioLegend), CD45 BV785 1:400 (30-F11, BioLegend), CD49b APC 1:100 (DX5, BD Pharmingen), CD90.2 APC-Cy7 1:400 (30-H12, BioLegend), CD117 PE 1:800 (2B8, eBioscience), CD117 APC 1:800 (2B8, BD Pharmingen), CD117 BV711 1:800 (2B8, BioLegend), FcεRI APC 1:200 (MAR-1, eBioscience), Gr-1 BV421 1:800 (RB6-8C5, BioLegend), Gr-1 BV605 1:200 (RB6-8C5, BioLegend), IgE PE 1:100 (RME1, BioLegend), IgE BV786 1:100 (RME-1, BD Pharmingen), IgE BV421 1:100 (RME-1, BD Pharmingen), Ly6G PerCP-Cy5.5 1:100 (1A8, BD Pharmingen), MHCII A700 1:100 (M5/114.15.2, eBioscience), Siglec-F BV421 1:100 (E50-2440, BD Pharmingen), Siglec-F PE 1:100 (E50-2440, BD Pharmingen), Ter119 BV421 1:200 (Ter119, BioLegend), 5-HT unconjugated 0.11 μg ml⁻¹ (5HT-H209, Dako) and mouse-IgG1 PE 1:100 (RMG1-1, BioLegend).

The viability and activation of 18 h MoS₂-treated cells were assessed using flow cytometry (Beckman Coulter Gallios). The cells were washed with 2% FBS in PBS (Flow Cytometry Staining Buffer, FACS Buffer), then stained with the respective antibody mix at 4 °C for 20 min. The anti-human antibodies used to characterize mast cells or to measure activation were FcεRI-FITC (Biolegend, #334608), CD117-APC (BD, #553356), MRGX2-PE (Biolegend, #359004), CD203c-PerCP/Cyanine5.5 (Biolegend, #324608), CD63-PE (BD, #353004) and CD107a-Alexa 647 (Biolegend, #328612). The viability of cells was analyzed by staining with Fixable Viability Dye (eBioscience FVD-eFluor 780, #65-0865-14). Reactive oxygen species (ROS) production was analyzed by staining with CM-H2DCFDA (Thermo Fisher Science, #C6827) for 30 min at 37 °C. After staining, the cells were washed twice with FACS buffer, then resuspended in fresh FACS buffer and analyzed on the flow cytometer as indicated above.

For immunoflourescent staining of tissue sections the following primary antibodies were employed: CD68, 1 µg/ml (clone: PGM-1, Dako Cytomation, Hamburg, Germany); CD14, 1 µg/ml (clone: TÜK4, Dako); CD86, 10 µg/ml (clone: FUN-1, BD Pharmingen, Heidelberg, Germany). Isotype- and concentration-matched control antibodies (Dako) served as negative controls.
For flow cytometric analysis the following mAb were purchased from BD Bioscience (Heidelberg, Germany): CD3 V450, CD14 FITC, CD33 PE-Cy7, CD86 AF700, HLA-DR V500, CD117 APC.

Pluripotency analysis was performed with the BD Stemflow™ Human and Mouse Pluripotent Stem Cell Analysis Kit (BD Biosciences Oxford, UK). Specific surface markers were assessed with fluorochrome-labelled specific antibodies after blocking Fc non-specific interactions by incubation with an anti-CD16/32 antibody for 10–15 minutes. Parameters for analysis were set with unlabelled cells and gating with isotype controls for the appropriate fluorochromes. Compensation for spectral overlap was done when required using BD™ CompBead Plus positive and negative beads. Analysis was run on a LSFORTESSA BD cytometer using Flowjo (7.6.5). Antibodies: CD71-FITC; CD117-APC; CD41-FITC; CD33-PE (BD pharmingen); CD61-PE (Caltag); CD45-Pacblue (biolegend); CD34-FITC (Cambridge Bioscience); CD14-APC (ebioscience)