Journal: The Journal of Immunology Author Choice

Article Title: Novel, Non–Gene-Destructive Knock-In Reporter Mice Refute the Concept of Monoallelic Gata3 Expression

doi: 10.4049/jimmunol.2000025

Figure Lengend Snippet: Noninvasive monitoring of Th2 cell polarization. (A) Flow cytometric analysis of Gata3-vYFP reporter expression (first column) and of intracellular GATA-3 protein levels (second column) in splenic CD4+ T cell populations from homozygous GATIR (KI/KI) and C57BL/6 control (wild-type [WT]/WT) mice before (Th0) and 6 d after in vitro polarization into Th1, Th2, and TH17 cells, respectively. Intracellular (ic) stainings for IFN-γ, IL-13, and IL-17A were used to confirm efficient polarization. Data are representative of three independent experiments. (B) Quantification of Gata3 transcript levels in Th cell populations before (Th0) and 6 d after in vitro polarization into Th1, Th2, or Th17 cells, respectively (top panel). Concomitant analysis of the same RNA samples for CD3e expression served as quality control for the presence of template RNA (lower panel). Data were normalized for β-actin expression, and values obtained with RNA from wild-type Th0 cells were arbitrarily set as 1 (dashed lines). RNA from FACS-sorted B cells served as control for primer specificity in both RT-qPCR analyses. RNA samples were analyzed in triplicate. Data are derived from a single polarization experiment distinct from the one shown in (A).

Article Snippet: Polarization experiments were performed in triplicate with 2.5 × 10 5 cells per well in 96-well plates precoated with 5 μg/μl anti-CD3 (catalog no. 100302; BioLegend) and 5 μg/μl anti-CD28 (catalog no. 102102; BioLegend), Abs in TexMACS Medium (catalog no. 130-097-196; Miltenyi Biotec) supplemented with 10% FBS and 0.01mM 2-ME (catalog no. 31350-010; Life Technologies), 1× penicillin–streptomycin (catalog no. 15140; Life Technologies), and various cytokines to induce polarization following the protocols of CytoBox Th1 mouse (catalog no. 130-107-761; Miltenyi Biotec), Th2 (catalog no. 130-107-760), and Th17 (catalog no. 130-107-758).

Techniques: Expressing, Control, In Vitro, Quantitative RT-PCR, Derivative Assay

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function.

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: Fig. 2. α2B/2C agonism suppresses chronic corneal epitheliopathy in dry eye disease (DED) and restores immunohomeostasis. (A) Treatment with the α2B/ 2C agonist (AGN-762) after the development of chronic DED effectively ameliorates the disease, assessed by corneal fluorescein staining (CFS) score. ****, p < 0.0001 vs untreated or vehicle group; n = 30 eyes (15 mice) /group from two independent experiments. (B) Corneal inflammation was assessed at the end of treatment by quantifying mature MHC-II+CD11b+ cells using flow cytometry and protein levels of IL-1β and IL-6 using ELISA. The upper panel shows the gating strategy for flow cytometry analysis. (C) Conjunctival inflammation was assessed by quantifying infiltrating CD4+ T cells using flow cytometry and protein levels of IL-17A, IL-17F, and IFN-γ using ELISA. The upper panel of the flow plots shows the gating strategy for flow cytometry analysis. (D) Draining lymph node (DLN) Th17 response was determined by flow cytometry. The upper panel shows the gating strategy for flow cytometry analysis. (E) The suppressive function of Treg cells on cell proliferation was quantified using the BrdU incorporation assay and compared with the proliferative responses in the absence of Treg (0 % suppression). For flow cytometry of corneal and conjunctival tissues, samples were pooled from 5 eyes in each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; data shown were from one representative experiment out of two performed and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Article Snippet: In vitro Th17 cell polarization Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, BrdU Incorporation Assay

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function.

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: Fig. 3. α2B/2C agonism provides sustained amelioration of corneal epitheliopathy and suppresses disease exacerbation. (A) A 7-day treatment with the α2B/2C agonist (AGN-762) in chronic DED (day 21 – 28) effectively maintains therapeutic efficacy for months (day 28 – 56), and suppresses disease exacerbation in treated animals upon re-challenged with desiccating stress (day 56 – 63). Disease severity was assessed by corneal fluorescein staining (CFS) score. ***, p < 0.001; ****, p < 0.0001 vs untreated or vehicle group; n = 20 eyes (10 mice) /group from two independent experiments. (B) Corneal inflammation was assessed at day 63 by quantifying mature MHC-II+CD11b+ cells using flow cytometry and protein levels of IL-1β and IL-6 using ELISA. The same gating strategy for flow cytometry analysis was used as shown in Fig. 2B. (C) Conjunctival inflammation was assessed at day 63 by quantifying protein levels of IL-17A, IL-17F, and IFN-γ using ELISA. (D) Draining lymph node Th17 response was determined by flow cytometry at day 63. The same gating strategy for flow cytometry analysis was used as shown in Fig. 2D. (E) The suppressive function of Treg cells on cell proliferation was quantified using the BrdU incorporation assay and compared with the proliferative responses in the absence of Treg (0 % suppression). For flow cytometry of corneal and conjunctival tissues, samples were pooled from 5 eyes in each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., not significant; data shown were from one representative experiment out of two performed and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Article Snippet: In vitro Th17 cell polarization Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Drug discovery, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, BrdU Incorporation Assay

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function.

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: Fig. 5. α2B/2C agonism (by AGN-762 at the higher concentration) enhances DED-derived effector T cells (Teff) in vitro. (A) Flow cytometry plots show the purity of freshly sorted Teff cells defined as CD4+CD25-Foxp3- cells that were subjected to cell cultures in vitro. (B) Flow cytometric analysis of CD4+CD25- Teff after 24 h culturing with AGN-762, with representative dot plots shown (the upper panel shows the gating strategy), and frequencies of IL-17A+IFN-γ- Th17, IL-17A+IFN- γ+ Th17/1, and IL-17A-IFN-γ+ Th1 summarized in the bar graphs. (C) ELISA assay on the culture supernatants for the levels of IL-17A and IFN-γ. Data shown were

Article Snippet: In vitro Th17 cell polarization Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Concentration Assay, Derivative Assay, In Vitro, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function.

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: Fig. 6. Adoptive transfer of ex vivo AGN-762-treated DED-Treg effectively reduces corneal epitheliopathy severity and Th17 immunity. (A) Treg cells were isolated from DED mice and in vitro cultured with 1 μM AGN-762 or vehicle for 24 h. The freshly sorted effector CD4+ T cells from DED (Teff) along with normal Treg, DED-Treg pre-treated with the vehicle, or DED-Treg pre-treated with AGN-762 were adoptively transferred to Rag1-/- mice, which were subsequently exposed to desiccating stress for 6 days. Disease severity was assessed by corneal fluorescein staining (CFS) score, and summarized from two independent experiments. n = 10–14 eyes (5–7 mice) /group. (B) Flow cytometric analysis of T cell response in the draining lymph nodes of Rag1-/- recipients at day 6, with representative dot plots shown on the left (the upper panel shows the gating strategy) and frequencies of IL-17-producing CD4 + T cells (including both IL-17A+IFN-γ- Th17 and IL-17A+IFN- γ+ Th17/1) summarized from two independent experiments on the right. *, p < 0.05; **, p < 0.01; data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Article Snippet: In vitro Th17 cell polarization Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Adoptive Transfer Assay, Ex Vivo, Isolation, In Vitro, Cell Culture, Staining

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function.

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: Fig. 7. Neutralization of IL-10 abrogates AGN-762-mediated restoration of Treg immunosuppressive function. (A) DED-Treg cells were pre-treated with 1 μM AGN-762 or vehicle for 24 h and then subjected to suppressive function assay in the presence of anti-IL-10 neutralizing antibody or isotype IgG control. (B) Treg cells were isolated from DED mice and in vitro cultured with 1 μM AGN-762 for 24 h. The freshly sorted effector CD4+ T cells from DED (Teff) along with DED-Treg pre- treated with AGN-762 were adoptively transferred to Rag1-/- mice, which were subsequently exposed to desiccating stress for 6 days. The recipients received treatment with anti-IL-10 antibody or control IgG immediately after adoptive transfer (day 0) as well as at day 3. Disease severity was assessed by corneal fluorescein staining (CFS) score, and summarized from two independent experiments. n = 16 eyes (8 mice) /group (C) Flow cytometric analysis of T cell response in the draining lymph nodes of Rag1-/- recipients at day 6, with representative dot plots shown on the left and frequencies of total T cells and IL-17-producing CD4 + T cells (including both IL-17A+IFN-γ- Th17 and IL-17A+IFN-γ+ Th17/1) summarized from two independent experiments on the right. *, p < 0.05; **, p < 0.01; data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test (A) or unpaired t test (B and C).

Article Snippet: In vitro Th17 cell polarization Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Neutralization, Functional Assay, Control, Isolation, In Vitro, Cell Culture, Adoptive Transfer Assay, Staining

Journal: Neuro-Oncology

Article Title: Agonist OX40 immunotherapy improves survival in glioma-bearing mice and is complementary with vaccination with irradiated GM-CSF–expressing tumor cells

doi: 10.1093/neuonc/nox125

Figure Lengend Snippet: Th1 vs Th2 BILs. (A) Flow cytometry demonstrating percent expression of T-bet, IFN-γ, and IL-12R β2 on CD4+ lymphocytes 18 days after GL261 tumor implantation by treatment group. Combination immunotherapy is associated with significant increases in all 3 Th1 markers. (***P < 0.0005, **P < 0.005, *P < 0.05; absence of an asterisk denotes not significant comparison.) (B) Flow cytometry demonstrating percent expression of GATA3, the IL-4 receptor, and IL-5 on CD4+ lymphocytes 18 days after GL261 tumor implantation by treatment group. Combination immunotherapy is associated with significant decreases in all 3 Th2 markers. (**P < 0.005, *P < 0.05, absence of an asterisk denotes not significant comparison.)

Article Snippet: For the analysis of T helper cell subsets, we used Multi-Color Flow Cytometry Kits (R&D Systems) for mouse Th1 cells (conjugated antibodies to T-bet-PerCP, IFN-gamma-fluorescein, IL-12 R beta 2-APC, and CD4-PE) and Th2 cells (conjugated antibodies to CD4-PerCP, IL-4 R-fluorescein, STAT6-APC, and IL-5-PE).

Techniques: Flow Cytometry, Expressing, Tumor Implantation, Comparison

Journal: Neuro-Oncology

Article Title: Agonist OX40 immunotherapy improves survival in glioma-bearing mice and is complementary with vaccination with irradiated GM-CSF–expressing tumor cells

doi: 10.1093/neuonc/nox125

Figure Lengend Snippet: Day 18 flow cytometry of BILs shows that vaccination improves the intratumoral CD8+/FoxP3+ lymphocyte ratio, which is little affected by treatment with anti-OX40 immunotherapy. (**P < 0.005, *P < 0.05; absence of an asterisk denotes not significant comparison.)

Article Snippet: For the analysis of T helper cell subsets, we used Multi-Color Flow Cytometry Kits (R&D Systems) for mouse Th1 cells (conjugated antibodies to T-bet-PerCP, IFN-gamma-fluorescein, IL-12 R beta 2-APC, and CD4-PE) and Th2 cells (conjugated antibodies to CD4-PerCP, IL-4 R-fluorescein, STAT6-APC, and IL-5-PE).

Techniques: Flow Cytometry, Comparison

Journal: BMC Nephrology

Article Title: Epoetin beta pegol for treatment of anemia ameliorates deterioration of erythrocyte quality associated with chronic kidney disease

doi: 10.1186/s12882-018-0818-4

Figure Lengend Snippet: Time course of Hb and RBC count. Hb levels ( a ) and RBC numbers ( b ) progressively and significantly decreased in the UNX-Thy1 + Vehicle group compared with levels in the Sham group. a C.E.R.A. treatment in the UNX-Thy1 group significantly increased Hb to levels nearly the same as those in the Sham group. b RBC number was also significantly increased by C.E.R.A. treatment in the UNX-Thy1 group. Values are shown as mean ± SEM. *, P < 0.05 ( a , b , two-way ANOVA followed by Tukey’s test). Sham, n = 5; UNX-Thy1 + Vehicle, n = 6; UNX-Thy1 + C.E.R.A., n = 6

Article Snippet: Briefly, after left kidney nephrectomy, an anti-rat CD90 (Thy1.1) monoclonal antibody (Cedarlane Labs, Burlington, Canada) at a single dose of 0.6 mg/kg body weight was intravenously injected into rats (UNX-Thy1 group, n = 12).

Techniques:

Journal: BMC Nephrology

Article Title: Epoetin beta pegol for treatment of anemia ameliorates deterioration of erythrocyte quality associated with chronic kidney disease

doi: 10.1186/s12882-018-0818-4

Figure Lengend Snippet: Plasma oxidative stress. The level of d-ROMs was significantly increased in the plasma of the UNX-Thy1 + Vehicle group compared with the Sham group. C.E.R.A. treatment in the UNX-Thy1 group tended to decrease the levels of plasma d-ROMs. Values are shown as mean ± SEM. *, P < 0.05 (Tukey’s test). Sham, n = 5; UNX-Thy1 + Vehicle, n = 6; UNX-Thy1 + C.E.R.A., n = 6

Article Snippet: Briefly, after left kidney nephrectomy, an anti-rat CD90 (Thy1.1) monoclonal antibody (Cedarlane Labs, Burlington, Canada) at a single dose of 0.6 mg/kg body weight was intravenously injected into rats (UNX-Thy1 group, n = 12).

Techniques: Clinical Proteomics

Journal: BMC Nephrology

Article Title: Epoetin beta pegol for treatment of anemia ameliorates deterioration of erythrocyte quality associated with chronic kidney disease

doi: 10.1186/s12882-018-0818-4

Figure Lengend Snippet: Erythrocyte deformability. Erythrocyte deformability is shown as the value of the elongation index. a Compared with the stable erythrocyte deformability status in the Sham group, erythrocytes in the UNX-Thy1 + Vehicle group showed a significant deterioration of deformability. C.E.R.A. treatment in UNX-Thy1 group significantly improved erythrocyte deformability. b A significant negative correlation was observed between MCV and erythrocyte deformability. Values are shown as mean ± SEM. *, P < 0.05 ( a , two-way ANOVA followed by Tukey’s test; b , Pearson’s correlation test). Sham, n = 5; UNX-Thy1 + Vehicle, n = 6; UNX-Thy1 + C.E.R.A., n = 6

Article Snippet: Briefly, after left kidney nephrectomy, an anti-rat CD90 (Thy1.1) monoclonal antibody (Cedarlane Labs, Burlington, Canada) at a single dose of 0.6 mg/kg body weight was intravenously injected into rats (UNX-Thy1 group, n = 12).

Techniques:

Journal: BMC Nephrology

Article Title: Epoetin beta pegol for treatment of anemia ameliorates deterioration of erythrocyte quality associated with chronic kidney disease

doi: 10.1186/s12882-018-0818-4

Figure Lengend Snippet: Erythrocyte turnover. a Biotinylated erythrocytes significantly decreased in the UNX-Thy1 + Vehicle group compared with in the Sham group. C.E.R.A. treatment in the UNX-Thy1 group significantly suppressed the decrease in biotinylated erythrocytes in circulation. b Although there was no statistical significance, total bilirubin in the plasma of the UNX-Thy1 + Vehicle group tended to increase compared with that in the Sham group. C.E.R.A. treatment in the UNX-Thy1 group tended to decrease plasma total bilirubin. Values are shown as mean ± SEM. *, P < 0.05 ( a , two-way ANOVA followed by Tukey’s test; b , Tukey’s test). Sham, n = 5; UNX-Thy1 + Vehicle, n = 6; UNX-Thy1 + C.E.R.A., n = 6

Article Snippet: Briefly, after left kidney nephrectomy, an anti-rat CD90 (Thy1.1) monoclonal antibody (Cedarlane Labs, Burlington, Canada) at a single dose of 0.6 mg/kg body weight was intravenously injected into rats (UNX-Thy1 group, n = 12).

Techniques: Clinical Proteomics

Journal: BMC Nephrology

Article Title: Epoetin beta pegol for treatment of anemia ameliorates deterioration of erythrocyte quality associated with chronic kidney disease

doi: 10.1186/s12882-018-0818-4

Figure Lengend Snippet: Intracellular calcium level in erythrocytes. a Intracellular calcium level as assessed by Fluo-3/AM intensity was significantly increased in erythrocytes in the UNX-Thy1 + Vehicle group compared with those in the Sham group. C.E.R.A. treatment in the UNX-Thy1 group significantly decreased intracellular calcium level to nearly the same level as in the Sham group. b A significant negative correlation was observed between intracellular calcium level in erythrocytes and erythrocyte deformability. Values are shown as mean ± SEM. *, P < 0.05 ( a , Tukey’s test; b , Pearson’s correlation test). Sham, n = 5; UNX-Thy1 + Vehicle, n = 6; UNX-Thy1 + C.E.R.A., n = 6

Article Snippet: Briefly, after left kidney nephrectomy, an anti-rat CD90 (Thy1.1) monoclonal antibody (Cedarlane Labs, Burlington, Canada) at a single dose of 0.6 mg/kg body weight was intravenously injected into rats (UNX-Thy1 group, n = 12).

Techniques:

Journal: Stem Cell Research

Article Title: Derivation of human induced pluripotent stem cell line EURACi004-A from skin fibroblasts of a patient with Arrhythmogenic Cardiomyopathy carrying the heterozygous PKP2 mutation c.2569_3018del50

doi: 10.1016/j.scr.2018.09.003

Figure Lengend Snippet: Characterization and validation.

Article Snippet: SOX2 and SSEA4 expression in iPSCs adapted to feeder-free condition was analyzed by flow cytometry using Multi-Color Flow Cytometry Kit (R&D System) following manufacturer's instruction.

Techniques: Biomarker Discovery, Light Microscopy, Immunocytochemistry, Expressing, Flow Cytometry, Gene Expression, Mutagenesis, Sequencing, Southern Blot

Journal: Stem Cell Research

Article Title: Derivation of human induced pluripotent stem cell line EURACi004-A from skin fibroblasts of a patient with Arrhythmogenic Cardiomyopathy carrying the heterozygous PKP2 mutation c.2569_3018del50

doi: 10.1016/j.scr.2018.09.003

Figure Lengend Snippet: Reagents details.

Article Snippet: SOX2 and SSEA4 expression in iPSCs adapted to feeder-free condition was analyzed by flow cytometry using Multi-Color Flow Cytometry Kit (R&D System) following manufacturer's instruction.

Techniques: Immunocytochemistry, Flow Cytometry, Control