il-35 human kit  (MyBiosource Biotechnology)

Journal: Advanced Science

Article Title: Tumor‐Associated Glycan Exploits Adenosine Receptor 2A Signaling to Facilitate Immune Evasion

doi: 10.1002/advs.202416501

Figure Lengend Snippet: GHCer promotes the differentiation of Treg cells and enhances its immunosuppressive activity. a) GHCer increases Treg differentiation from T cells of healthy donors. Human CD4 + T cells were cultured in a Treg differentiation medium containing anti‐CD3 and anti‐CD28 monoclonal antibodies, IL‐2, and TGF‐β in the presence of 30 µM GHCer, SSEA3Cer, SSEA4Cer, or PBS for 6 days. CD4 + T cells cultured in PBS for 6 days without activation by anti‐CD3 and anti‐CD28 antibodies were used as a control for Treg differentiation. b) GHCer‐induced Treg cells exhibit greater suppression of the proliferation of Tconv cells. Suppressive activities of human CD4 + CD25 + Treg induced in the presence of GHCer or PBS after washing twice were assessed by incubating with CFSE‐labelled, anti‐CD3/CD28 activated CD4 + CD25 − T cells (Tconv) at the indicated ratios. At 72 h, the proliferation of Tconv was analyzed by FACS. The Tconv cells without Treg cells were used as a control for normalization, represented by 100% (a black circle). The percentage of proliferating cells was normalized against the Tconv cells only. c) IL‐35 in the supernatants from GHCer‐induced Treg cells was measured using an IL‐35 Sandwich ELISA. d) IL‐10 production in supernatants from GHCer‐induced Treg cells was measured using an IL‐10 Sandwich ELISA. e) Flow cytometry analysis of LAG3, CTLA‐4, PD‐L1, CD39, and CD73 expression on human Treg cells induced in PBS or GHCer. f) Adenosine concentration in the supernatants of differentiated Treg on day 6. Supernatants from Treg differentiation were filtered through a 0.22 µm filter. Adenosine in the supernatant was measured by colorimetric assay. Data represent three experiments, and values are expressed as means ± SD. Statistical significance was calculated using ANOVA with Tukey correction for multiple comparisons. ∗p < 0.05; ∗∗p < 0.01; ***p < 0.001.

Article Snippet: IL‐10 and IL‐35 concentrations were measured in the supernatants of Treg differentiation cultures after 6 days, as described above, using specific ELISA kits (human: CSB‐ E13126 h, CUSABIO; mouse: 440507, BioLegend) according to the manufacturer's instructions.

Techniques: Activity Assay, Cell Culture, Bioprocessing, Activation Assay, Control, Sandwich ELISA, Flow Cytometry, Expressing, Concentration Assay, Colorimetric Assay

Journal: PeerJ

Article Title: Malignant behaviors and immune response in melanoma: Epstein-Barr virus induced gene 3 as a therapeutic target based on an in-vitro exploration

doi: 10.7717/peerj.18730

Figure Lengend Snippet: (A–C) Quantification on the phosphorylation of SMAD3 and STAT6 in EBI3 -silenced melanoma cells MV3 via western blotting. (D) Measurement on the immune response-related cytokines IL-35, IL-4 and IL-13 in EBI3 -silenced melanoma cells MV3 via ELISA. * p < 0.05; ** p < 0.01.

Article Snippet: In this study, melanoma cells were cultured in the six-well culture plates at the density of 8 × 10 5 /well for 24 h. Hereafter, the cell culture supernatant was harvested for the calculation on the levels of relevant cytokines including IL-35 (NBP3-06774), IL-4 (NBP1-91171) and IL-13 (NBP1-91176) as per the manuals of the producer (Novus Biologicals, Centennial, CO, USA).

Techniques: Phospho-proteomics, Western Blot, Enzyme-linked Immunosorbent Assay