Golgistop kit

To confirm the engraftment of human immune cells in mouse blood, flow cytometry was performed. To confirm the engraftment of human CD4 T cells, mononuclear cells in mouse blood were stained with human anti-CD4-PeCy7 (Cat. no. 300511; Biolegend, San Diego, CA). Four weeks after tissue transplantation, IL-17-expressing human CD4⁺ T cells (Th17) were analyzed in the whole blood of mice. Mononuclear cells in mouse blood were stained with human anti-CD4 and IL-17-PE (12-7179-42; eBioscience, San Diego, CA). Before intracellular staining, the cells were stimulated for 4 h with phorbol myristate acetate (25 ng/mL) and ionomycin (250 ng/mL) in the presence of GolgiStop (554715; BD Biosciences, San Jose, CA). Intracellular staining was performed using a BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit and BD GolgiStop Kit (554715; BD Biosciences). Flow cytometry was performed using a cytoFLEX flow cytometer (Beckman Coulter, Brea, CA), and the data was analyzed using FlowJo software (Tree Star, Ashland, OR).

Mouse lymphocytes were stained with following fluorochrome conjugated antibodies: CD4(L3T4)-PerCP Cy5.5, CD25(PC61)-APC, Foxp3(FJK-16 s)-PE, IFN-γ(XMG1.2)-APC, IL-17(eBio17B7)-FITC, B220(RA3-6B2)-APC, CD19(eBio1D3(1D3))-PerCP, CD1d(1B1)- PE, CD5(53–7.3)-FITC, IL-10(JES5-16E3)-APC, CD138(281–2)-PE, and T- and B-Cell Activation Antigen (GL7)-FITC. Human lymphocytes were stained with following fluorochrome conjugated antibodies: CD4(RPA-T4)-PECy7, CD25(BC96)-APC, Foxp3(259D/C7)-PE, IFN-γ(4S.B3)-APC, IL-17(eBio64DEC1-FITC, CD19(HIB19)-FITC, IL-10(JES3-19F1)-APC, CD138(MI15)-PB450. Before intracellular staining, cells were stimulated for 4 h with phorbol myristate acetate (25 ng/mL) and ionomycin (250 ng/mL) in the presence of Golgistop (BD Biosciences, San Jose, CA, USA). Intracellular staining was fixed using a BD Cytofix/ Cytoperm Plus Fixation/Permeabilization Kit and BD Golgistop Kit (BD Biosciences, San Jose, CA). Foxp3, transcription factor was fixed using a Foxp3/Transcription Factor Staining buffer set (eBioscience, San Diego, CA) following the manufacturer’s instructions. Flow cytometric analysis was performed using a cytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, USA) and collected data were analyzed using the FlowJo software (Tree Star, Ashland, OR).

BM cells were differentiated into cDC1-like cells and incubated for 4 h in 96-well U-bottom plates (5 × 10⁴ per well) in 200 µl of medium in the presence of 2 mg OVA protein (10 mg ml^–1; vac-stova; InvivoGen) and individual interleukins (IL-2, IL-12, IL-15, IL-18, IL-21, IL-23 or IL-27). The concentration of each interleukin was 2 ng ml^–1, 8 ng ml^–1 or 20 ng ml^–1 (in 200 µl). Meanwhile, OT-I CD8⁺ and OT-II CD4⁺ cells were isolated using EasySep kits (STEMCELL Technologies) and resuspended in T cell medium (complete RPMI medium with 50 μM beta-mercaptoethanol, minimal non-essential amino acids, 1 mM sodium pyruvate and 10 mM HEPES). OVA-loaded cDC1-like cells were then washed and co-cultured with OT-I or OT-II cells in T cell medium in the presence of the aforementioned interleukins. Co-culture of OVA-loaded cDC1-like cells with OT-I or OT-II cells was continued for 3 days and 5 days, respectively. T cell activation was measured by intracellular staining with antibodies against IFNγ (clone XMG1.1, BD Biosciences) using BD Golgi Stop kit (BD Biosciences).

Mouse lymphocytes were immunostained using fluorescently conjugated antibodies against CD4, IL-17, IL-10, and IL-4. For intracellular staining, cells were first stimulated for 4 h with phorbol myristate acetate (25 ng/mL) and ionomycin (250 ng/mL) in the presence of BD GolgiStop (BD Biosciences, San Jose, CA, USA). Intracellular staining was performed using a BD Cytofix/CytopermPlus Fixation/Permeabilization Kit and BD GolgiStop Kit (BD Biosciences). Flow cytometry was performed using a cytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, USA).

The mesenteric lymph nodes (MLNs) were removed from the mice and single cells were isolated and immunostained using fluorescently conjugated antibodies against CD4, IL-17, CD25, Foxp3, CD19, CD5, CD1d, and IL-10. Prior to intracellular staining, cells were stimulated for 4 h with phorbol 12-myristate 13-acetate (PMA) (25 ng/mL) and ionomycin (250 ng/mL) in the presence of GolgiStop™ (BD Biosciences, USA). Intracellular staining was performed using a BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit and BD Golgistop Kit (BD Biosciences, USA). The transcription factor Foxp3 was stained using a Foxp3/Transcription Factor Staining Kit (eBioscience, USA) according to the manufacturer’s instructions.
PBMCs from patients with UC were restimulated with PMA (25 ng/mL) and ionomycin (250 ng/mL) in the presence of GolgiStop™ for 4 h and immunostained for CD4, IL-17, CD25, and Foxp3 (eBiosciences, USA). Flow cytometry was performed using a cytoFLEX Flow Cytometer (Beckman Coulter, USA).