Cd25 apc

After 5-day incubation, cells were stained with anti-CD8 PerCP-Cy5.5, CD4 PE, CD25 APC and anti-BrdU FITC (BD, Franklin Lakes, NJ, USA); while regulatory T cells (Treg) were identified with anti-CD4 PE, FoxP3 PerCP-Cy5.5, CD25 APC (BD, Franklin Lakes, NJ, USA). Samples were analyzed by flow cytometry. 10,000 events were registered within the lymphocyte region identified according to their size (Forward-scatter, FCS) and complexity (Side-scatter, SSC), considering their activation status [46 (link)].
Data were analyzed with FlowJo software (v.10; Tree Star, Inc., Ashland, OR, USA).

To determine the immune cell profile of whole polyp cells, fresh and cultured (for 72 hours) cells were harvested (both adherent and nonadherent cells) without enzymatic dissociation and labeled with a set of various specific fluorescent antibodies, such as CD4-Pacifc Blue, CD8-PECy7, CD11c-PE, CD14-PerCPCy5.5, CD19-Viollet, CD25-APC, CD40-FITC, CD69-FITC, and NK1.1-PE (all from BD Biosciences). These sets of antibodies were used for both fresh and in vitro coculture analysis. To investigate the CD4+CD25+Foxp3+ T lymphocyte profile, intracellular staining for FoxP3 expression was performed after 72 hours in culture with or without the presence of MSCs. The cells were fixed and permeabilized using the Fix/Perm Buffer Set Kit (BD Biosciences). Staining was performed using FoxP3 (PE), CD4 (APC-Cy7), and CD25 (APC) antibodies at 1 : 100 dilution (BD Biosciences). Analysis was determined by flow cytometry (FACSCanto II cytometer, BD Biosciences) within FSC and SSC gate in CD4+ T cells. All acquisitions were performed using a FACSCanto II flow cytometer (BD Biosciences), and analysis was again done using the FlowJo 7.2.4 software (Tree Star). During analysis, gates for mononuclear cells were performed in FSC and SSC parameters after doublet exclusion. Results are presented as cell frequency.

Human IgD was purchased from Abcam (Cambridge, MA, USA). Anti-CD3 and CD28 antibodies were provided by T&L Biological Technology (Beijing, China). A770041 was purchased from Axon Medchem (Groningen, Netherlands). rhTNFR:Fc fusion protein was purchased from Guojian Pharmaceutical Company (Shanghai, China). Anti-mouse IgD antibodies were purchased from eBioscience (San Diego, CA, USA). Anti-human CD69-PE-cy5, CD154-PE, CD4-FITC, CD25-APC, IL-4-APC, IFN-γ-PE-cy7, IL-17-PE, and FoxP3-PE, along with anti-mouse CD3e-PerCP-Cy^TM5.5, CD4-FITC, CD25-APC, CD154-PE, IFN-γ-PerCP-Cy^TM5.5, IL4-APC, IL-17-PE, and FoxP3-PE antibodies were provided by BD Pharmingen (San Diego, CA, USA). Anti-Lck, anti-phospho-ZAP70, anti-phospho-Lck, anti-ZAP70, and anti-β-actin were purchased from Affinity Biosciences, Sigma and Abcam.