antibody pe-conjugated anti-mouse cd25 (pc61.5)  (Thermo Fisher)

Journal: eLife

Article Title: Thymic dendritic cell-derived IL-27p28 promotes the establishment of functional bias against IFN-γ production in newly generated CD4 + T cells through STAT1-related epigenetic mechanisms

doi: 10.7554/eLife.96868

Figure Lengend Snippet: ( A ) CD4SP (GFP + CD4 + CD8 - CD44 lo ) thymocytes, CD4 + RTEs (GFP + CD4 + CD8 - CD25 - CD44 lo ) and CD4 + naive (GFP - CD4 + CD8 - CD25 - CD44 lo ) T cells were sorted from 6- to 8-week-old Cd11c-p28 f/f mice and WT littermates, stimulated with plate coated anti-CD3 (2 µg/mL) and soluble anti-CD28 (1 µg/mL) for 12 hr. mRNA levels of Ifng, Il4, and Il2 were determined by qPCR. Data: mean ± SD (n=4, duplicates). ( B ) Sorted cells were cultured under Th0 conditions for 3 days. The frequency of IFN-γ-producing CD4 + T cells were measured by intracellular staining. Representative dot plots (left) and statistical data (right, mean ± SD, n=3). ( C ) Supernatants from 3-day cultures were analyzed for IFN-γ and IL-4 by ELISA. Data: mean ± SD (n=3). ( D ) mRNA levels of Tbx21 and Gata3 in sorted cells were determined by qPCR. Data: mean ± SD (n=4, duplicates). ( E–F ) T-bet protein levels were assessed by western blot ( E ) and flow cytometry ( F ) after 3-day culture. Data: mean ± SD (n=3). ( G ) Freshly sorted cells were lysed in Trizol, and Ifng and Tbx21 mRNA levels were determined by qPCR. Data: mean ± SD (n=4, duplicates). Statistical differences: * p <0.05, ** p <0.01, *** p <0.001 (Student’s t -test). Figure 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Antibody , PE- conjugated anti-mouse CD25 (PC61.5) , eBioscience , Cat#: 12-0251-82 RRID: AB_465607 , FACS (5 μL per test).

Techniques: Cell Culture, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Interferon-γ-stimulated antigen-presenting cancer-associated fibroblasts hinder neoadjuvant chemoimmunotherapy efficacy in lung cancer

doi: 10.1016/j.xcrm.2025.102017

Figure Lengend Snippet:

Article Snippet: CD25 Monoclonal Antibody (PC61.5), PE-Cyanine7 , Thermo Fisher Scientific , Cat# 25-0251-81; RRID: AB_469607.

Techniques: Recombinant, Staining, Purification, In Vivo, Control, Protease Inhibitor, Activation Assay, Membrane, Modification, Bicinchoninic Acid Protein Assay, Immunohistochemical staining, Enzyme-linked Immunosorbent Assay, Cell Isolation, Sequencing, Negative Control, shRNA, Software

Journal: Cell reports

Article Title: Vimentin modulates regulatory T cell receptor-ligand interactions at distal pole complex, leading to dysregulated host response to viral pneumonia

doi: 10.1016/j.celrep.2024.115056

Figure Lengend Snippet: (A) Weight change of Rag1 −/− mice following adoptive transfer of 4 × 10 5 Tcon cells ( n = 5) alone or with 0.5 × 10 5 WT or Vim −/− Tregs. Data are mean ± SEM. (B) H&E staining of distal colon from Rag1 −/− mice 45 days post-transfer. Scale bar, 100 μm. (C) Histological scores of colon tissue from (B). (D) Vimentin expression in WT and Vim −/− nTregs. Scale bar, 5 μm. (E) In vitro suppression assay by co-culturing Tcon cells with WT or Vim −/− nTregs, showing proliferating cell percentage (left) and suppression percentage (right) ( n = 3). Data are mean ± SD, n = 3. (F) Expression of Foxp3, CD25, Helios, and CD73 on CD4 + Foxp3 + cells from WT and Vim −/− spleens. Data are mean ± SD, n = 3–6 mice per group. p values were calculated via one-way ANOVA (C), two-way ANOVA (E), and two-tailed unpaired Student’s t test (F). NS, not significant ( p > 0.05), * p < 0.05, ** p < 0.005, and *** p < 0.0005.

Article Snippet: CD25 Monoclonal Antibody (PC61.5), PE, eBioscience , eBioscience , Cat#12–0251-81; RRID:AB_465607.

Techniques: Adoptive Transfer Assay, Staining, Expressing, In Vitro, Suppression Assay, Two Tailed Test

Journal: Cell reports

Article Title: Vimentin modulates regulatory T cell receptor-ligand interactions at distal pole complex, leading to dysregulated host response to viral pneumonia

doi: 10.1016/j.celrep.2024.115056

Figure Lengend Snippet: (A) Flow cytometry contour plots and quantification of IL-18R + Treg frequency among lung Tregs of WT and vimentin cKO mice after influenza infection. (B) IL-18R expression on lung Tregs after influenza infection ( n = 4–5 mice per group). (C) IL-18R + Treg number in whole lung after influenza infection. (D) BALF Areg levels measured by ELISA ( n = 3–8 mice per group). (E) Areg expression in lung Treg by qPCR at 10 dpi. (F) CD4 + CD25 + lung Tregs cultured with IL-2, CD3/CD28 beads, and 100 ng/mL IL-18 for 4 days, with Areg levels assessed in supernatant by ELISA. (G–I) WT and vimentin cKO mice infected with IAV; vimentin cKO mice received anti-Areg (50 μg/mouse, intraperitoneal) at 5 dpi, 4 times in 3 day intervals, or vehicle control. Lung histology at 21 dpi. Scale bar, 500 μm. (J and K) Representative contour plots (J) and Ki67 + IL-18R + Treg frequency (K) among lung Tregs after influenza infection. Data are mean ± SD, n = 3–5 mice per group. p values were calculated by two-way ANOVA (A–F and K) and two-way ANOVA (I). * p < 0.05, ** p < 0.005, *** p < 0.0005, and **** p < 0.0001.

Article Snippet: CD25 Monoclonal Antibody (PC61.5), PE, eBioscience , eBioscience , Cat#12–0251-81; RRID:AB_465607.

Techniques: Flow Cytometry, Infection, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Control

Journal: Cell reports

Article Title: Vimentin modulates regulatory T cell receptor-ligand interactions at distal pole complex, leading to dysregulated host response to viral pneumonia

doi: 10.1016/j.celrep.2024.115056

Figure Lengend Snippet: (A and B) Confocal images of vimentin and CD43 or ezrin in WT iTregs treated with DMSO (vehicle control) or WFA and Vim −/− iTregs. Scale bar, 5 μm. (C and D) Confocal images showing uniform IL-18R and CD73 distribution on vimentin-deficient nTregs. Scale bar, 5 μm. (E) Confocal images of vimentin and IL-18R in WT nTregs treated with vehicle control or 1 μM WFA for 24 h. Scale bar, 5 μm. (F) Vimentin expression in WT iTregs treated with 1 μM WFA or vehicle control for 24 h. Scale bar, 5 μm. (G) Carboxyfluorescein succinimidyl ester (CFSE) dilution in CD4 + CD25 − Tcon cells co-cultured with WT iTregs treated with WFA or vehicle control ( n = 3). (H) CD4 + CD25 + lung Tregs from WT mice were cultured with IL-2, CD3/CD28 beads, and 100 ng/mL IL-18 for 3 days, followed by 1 μM WFA treatment for 24 h. Supernatants were collected for Areg measurement by ELISA ( n = 6–7 per group). Data are presented as mean ± SD. p values were calculated by two-way ANOVA (G) and two-tailed unpaired Student’s t test (H). ** p < 0.005, *** p < 0.0005, and **** p < 0.0001.

Article Snippet: CD25 Monoclonal Antibody (PC61.5), PE, eBioscience , eBioscience , Cat#12–0251-81; RRID:AB_465607.

Techniques: Control, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cell reports

Article Title: Vimentin modulates regulatory T cell receptor-ligand interactions at distal pole complex, leading to dysregulated host response to viral pneumonia

doi: 10.1016/j.celrep.2024.115056

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD25 Monoclonal Antibody (PC61.5), PE, eBioscience , eBioscience , Cat#12–0251-81; RRID:AB_465607.

Techniques: Purification, Blocking Assay, Plasmid Preparation, Functional Assay, Control, Virus, Recombinant, Avidin-Biotin Assay, Cell Isolation, Enzyme-linked Immunosorbent Assay, Software

Journal: Frontiers in Oncology

Article Title: DSP-0509, a TLR7 agonist, exerted synergistic anti-tumor immunity combined with various immune therapies through modulating diverse immune cells in cancer microenvironment

doi: 10.3389/fonc.2024.1410373

Figure Lengend Snippet: DSP-0509 reduced Treg function and differentiation, and when combined with IDO1 inhibitor, enhanced anti-tumor activity. (A) Representative histogram of CellTrace™ Violet (CTV) fluorescence. CD4 Teff cells were co-cultured with Tregs for 3 days. CD4 Teff cell proliferation was evaluated by the ratio of CTV fluorescence intensity. (B) The proliferation index was calculated based on CTV fluorescence intensity. *P < 0.01 two-tailed Student t test vs control. Values are the mean ± S.E.M. of each group, n = 3/group. (C) Tregs were differentiated from CD4 + cells in the presence of TGFβ and CD11c + DCs. Tregs were detected as CD4 + CD25 + Foxp3 + cells. *P < 0.05 for the τ-test. (D) Curve of HM-1 tumor growth. DSP-0509 5 mg/kg i.v. was administered once weekly and Epacadostat 100 mg/kg p.o. was administered once daily. *P < 0.05 for the Dunnett test. n = 6/group and the values are the mean and S.E.M. (E) TILs were isolated from HM-1 tumor at day 21. Tregs and CD8 + cells were analyzed by flow cytometry. *P < 0.05 for the Wilcoxon rank sum test.

Article Snippet: Flow cytometry analysis was carried out using the following primary antibodies: PE-CD11b (BD Biosciences, M1/70), FITC-Gr-1 (BD Biosciences, RB6-8C5), FITC-CD8a (BD 53-6.7), FITC-CD4 (eBioscience, Inc., RM4-5), PE-CD25 (eBioscience, Inc., PC61.5), APC-CD45RB (eBioscience, Inc., FJK-16s), PE-Cy7-CD11b (BD Biosciences, M1/70), and PE-CD86 (BD Pharmingen, GL1), and the following viability dyes: Fixable Viability Stain 450 (BD Horizon) and Fixable Near-IR Dead Cell Stain (Invitrogen).

Techniques: Activity Assay, Fluorescence, Cell Culture, Two Tailed Test, Control, Isolation, Flow Cytometry