magcellect mouse cd4 cd25 regulatory t cell isolation kit  (R&D Systems)

Journal: Scientific Reports

Article Title: Genistein enhances anti-PD-1 efficacy in melanoma by suppressing regulatory T cell differentiation and activity

doi: 10.1038/s41598-025-20941-7

Figure Lengend Snippet: Genistein reversed the immunosuppressive microenvironment in melanoma. ( A ) Quantification of tumor-infiltrating CD3⁺CD8⁺ cytotoxic T lymphocytes (Gating Strategy Fig. ). ( B ) Quantification of tumor-infiltrating CD4 + Foxp3 + Tregs. ( C ) Ratio of CD8 + cytotoxic T cells to Tregs. ( D ) Concentrations of IFN-γ, TNF-α, and IL-10 in tumor tissues measured by ELISA. ( n = 5). Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cell suspension was filtered through a 40 μm cell strainer, and splenocytes were isolated using the MagCellect Mouse CD4 + CD25 + Regulatory T Cell Isolation Kit (MAGM208, R&D systems, CA, USA) to obtain primary CD4 + T lymphocytes and CD4 + CD25 + Tregs.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Genistein enhances anti-PD-1 efficacy in melanoma by suppressing regulatory T cell differentiation and activity

doi: 10.1038/s41598-025-20941-7

Figure Lengend Snippet: Genistein dose-dependently impaired Treg function. ( A ) RT-qPCR analysis of Treg functional markers (Foxp3, CTLA-4, and LAG-3) after 24-h treatment with genistein at concentrations of 0–20 µM. ( B ) Western blot detection of Foxp3 protein expression following 24-h exposure to genistein (0–20 µM). ( C ) ELISA quantification of IL-10 in Treg culture supernatants after 24-hour genistein treatment (0–20 µM). ( D ) CD4 + CD25 − Th cells (2 × 10 5 /ml) were labeled with CFSE and subsequently cocultured with Tregs preincubated with genistein (10 µM) for 48 h. The Th cell proliferation was determined by flow cytometry. ( E ) ELISA quantification of IFN-γ and TNF-α in supernatants from Treg and Th cell co-cultures. ( n = 3). Data are showed as mean ± SD; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cell suspension was filtered through a 40 μm cell strainer, and splenocytes were isolated using the MagCellect Mouse CD4 + CD25 + Regulatory T Cell Isolation Kit (MAGM208, R&D systems, CA, USA) to obtain primary CD4 + T lymphocytes and CD4 + CD25 + Tregs.

Techniques: Quantitative RT-PCR, Functional Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Labeling, Flow Cytometry

Journal: Scientific Reports

Article Title: Genistein enhances anti-PD-1 efficacy in melanoma by suppressing regulatory T cell differentiation and activity

doi: 10.1038/s41598-025-20941-7

Figure Lengend Snippet: Genistein suppressed Treg differentiation by targeting the PI3K-Akt pathway. ( A ) CD4 + CD25 − naïve T cells were differentiated into Tregs under anti-CD3/CD28 stimulation and 2 ng/ml TGF-β1 for 72 h, with or without 10 µM genistein. Flow cytometry was used to quantify CD4 + Foxp3 + cells. ( B ) qRT-PCR analysis of Treg signature genes ( Foxp3 , CTLA-4 , LAG-3 , IL-10 ) in cells treated as in ( A ). ( C ) Western blot detection of phosphorylated PI3K and AKT in the induced T cells, which were incubated with genistein (10 µM) or the combination of genistein (10 µM) and SC-79 (10 µM) under Treg-inducing conditions for 72 h. ( D ) Flow cytometric quantification of CD4 + Foxp3 + cells under the conditions in ( C ). ( E ) mRNA expression of Foxp3, CTLA-4, LAG-3 and IL-10 in induced T cells under the conditions in ( C ). Data are presented as mean ± SD ( n = 3); * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cell suspension was filtered through a 40 μm cell strainer, and splenocytes were isolated using the MagCellect Mouse CD4 + CD25 + Regulatory T Cell Isolation Kit (MAGM208, R&D systems, CA, USA) to obtain primary CD4 + T lymphocytes and CD4 + CD25 + Tregs.

Techniques: Flow Cytometry, Quantitative RT-PCR, Western Blot, Incubation, Expressing