Cd203c bv421 clone np4d6

The stromal vascular fraction was labeled with 2 μl of controls or fluorophore-conjugated antibodies in Eppendorf tubes at room temperature for 20 minutes. After that, the cells were fixed, and the erythrocytes were lysed with 1 ml of BD FACS Lysing Solution for 30 minutes. Then, the samples were centrifuged 10 minutes at 3500 x g, and the pellets were resuspended in 500 μl of PBS. Subsequently, the samples were stored at 4°C until the next day. Flow cytometry was performed using a FACS ARIA III equipment, and data were acquired on a logarithmic scale. The internal standard was used to calculate the number of cells per mg of tissue.
The fluorescent-conjugated antibodies used to identify mast cells were: anti-CD45 PE-CF594 (clone HI30, BD), anti-CD117 APC (clone YB5.B8, BD), anti-FcϵRI PE-Cy7 (clone AER-37, BioLegend), and CD203c BV421 (clone NP4D6, BioLegend). Additionally, the antibodies CD34 BV785 (clone 561, BioLegend) and integrin β7 FITC (clone FIB504, BioLegend) were used in a subcohort of 15 patients (6 non-T2D, 6 pre-T2D, and 3 T2D). Compensation beads and isotype controls were purchased from BD Biosciences. MC were identified as CD45⁺ CD117⁺ CD203c⁺ FcϵRIα⁺ (Figure 1A).

The resuspended stromal vascular fraction was stained with 2 μl of fluorophore-conjugated antibodies or controls in Eppendorf tubes for 20 minutes at room temperature. Later, samples were fixed, and erythrocytes were lysed adding 1 ml of BD FACS Lysing Solution for 30 minutes. After that, samples were centrifuged at 3500 x g for 10 minutes, and pellets were resuspended in 500 μl of PBS. Then, samples were stored until the next day at 4°C. A FACS ARIA III equipment was employed to perform the flow cytometry, and data were acquired on a logarithmic scale. The fluorescence of the internal standard was used to normalize the signal obtained from the fluorophore-conjugated antibodies (Supplementary Figures 1–4).
The fluorophore-conjugated antibodies employed were: anti-CD117 APC (clone YB5.B8, BD), anti-CD45 PE-CF594 (clone HI30, BD), anti-FcϵRI PE-Cy7 (clone AER-37, BioLegend), and CD203c BV421 (clone NP4D6, BioLegend). The voltages employed were APC (560V), PE-CF594 (430V), PE-Cy7 (530V), and BV421 (400V). Isotype controls and compensation beads were purchased from BD Biosciences. Mast Cells were identified as CD45⁺ CD117⁺ cells.