Alexa fluor 647 conjugated anti stat3 phospho tyr705

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 measure the proportion and differential subsets of Vγ9Vδ2 T lymphocytes, we performed surface staining with FITC-conjugated anti-CD3 (Tianjin Sungene Biotech Co., Ltd.), PerCP-conjugated anti-TCR Vδ2, Pacific Blue-conjugated anti-CD27 and APC-conjugated anti-CD45RA (BioLegend) antibodies. To evaluate the functional markers of Vγ9Vδ2 T lymphocytes, cells were restimulated with 50 ng/ml PMA and 500 ng/ml ionomycin for 6 h, permeabilized using reagents from a BD Cytofix/Cytoperm Kit and stained with PE-Cy7-conjugated anti-NKG2D, APC-conjugated anti-IFN-γ (BD Biosciences), PE-conjugated anti-Perforin and Pacific Blue-conjugated anti-Granzyme B (BioLegend) antibodies. GolgiStop (BD Biosciences) was added for the last 4 h of culture. To evaluate intracellular protein, DLBCL cells were sorted from the co-culture, fixed, permeabilized using BD Phosflow™ (BD Biosciences) and stained with Alexa Fluor® 647-conjugated anti-STAT3 Phospho (Tyr705) (Biolegend). To measure PD-L1⁺ or PD-1⁺ cells, Vγ9Vδ2 T lymphocytes were co-incubated with DLBCL cells for 6 h. We then performed surface staining with BV421-conjugated anti-PD-L1 (Biolegend) or Pacific Blue-conjugated anti-PD-1 (Biolegend) antibodies. For the cell survival assay, an Annexin V/PI apoptosis kit (Bimake) was used. The data were analyzed using FlowJo software (Tree Star, lnc).