Percp conjugated anti human cd45

Single-cell suspensions isolated from intestinal tissues of CD patients were stained with FITC-conjugated anti-human lineage cocktail 3 (Lin3) (CD3, CD14, CD19, and CD20; clone: MφP9, L27, SK7, and SJ25C1; BD Biosciences), PerCP-conjugated anti-human CD45 (clone: HI30, BioLegend), APC-conjugated anti-human CD127 (clone: A019D5, BioLegend), PE-conjugated anti-human NKp44 (clone: P44-8, BioLegend), PE-Cy7-conjugated anti-human CD117 (clone: 104D2, eBioscience), Brilliant Violet 421-conjugated anti-human CRTH2 (clone: BM16, BioLegend), and Alexa Fluor 647-conjugated anti-STAT3 Phospho-Tyr705 (clone: 13A3-1, BioLegend). Viability was assessed by Fixable Yellow Dead Cell Stain (Thermo Fisher Scientific). For phosphoflow staining, cells were stimulated with 20 ng/ml of recombinant human IL-23 (carrier free, R&D) for 15 mins and were permeabilized by True-Phos™ Perm Buffer (BioLegend) following the manufacturer's protocol. AbC™ Total Antibody Compensation Beads (Cat. No. A10497, Life Technologies) and ArC™ Amine Reactive Compensation Beads (Cat. No. A10346, Life Technologies) were used for compensation. Data were acquired on an LSRFortessa™ flow cytometer (BD Biosciences) using BD FACSDiva™ software in the Cytometry Core at the University of Florida and analyzed by FlowJo software (Version 10, Tree Star Inc.).

To determine the expression of mesenchymal stem cell markers on T-MSCs, cells were collected and pelleted by centrifugation at 1300 rpm for 5 min. After washing with PBS, cells were pelleted again and stained with FITC-conjugated anti-human CD11b, PE-conjugated anti-human CD34, PerCP-conjugated anti-human CD45, APC-conjugated anti-human CD73, PE-conjugated anti-human CD90, or PE-conjugated anti-human CD105 antibody (Biolegend, San Diego, CA, USA) diluted in FACS buffer (PBS supplemented with 10% FBS, 10 mM EDTA, 20 mM HEPES, and 1 mM sodium pyruvate) for 30 min on ice. To determine the expression of megakaryocytic marker on differentiated K562 cells, Pacific Blue-conjugated anti-human CD41 antibody (Biolegend) was used. After washing cells with buffer, surface protein expression was measured using a NovoCyte flow cytometer (ACEA Biosciences, San Diego, CA, USA) and analyzed using NovoExpress software.