Journal: Frontiers in Immunology

Article Title: Sphingosine-1-Phosphate (S-1P) Promotes Differentiation of Naive Macrophages and Enhances Protective Immunity Against Mycobacterium tuberculosis

doi: 10.3389/fimmu.2019.03085

Figure Lengend Snippet: Sphingosine-1-phosphate (S-1P) skews M1 effector response in macrophages. (A) RAW264.7 macrophages were stimulated as indicated, and NO titers were quantified from their culture supernatants. Shown here is the μM of NO ± SEM from three independent experiments. (B) Whole cell lysates of macrophages used above were collected 24 h post-treatment culture and analyzed for the expression of iNOS (M1 effector protein) and Sphk-1 proteins by immune blotting. A representative blot from several repeats with similar outcome is shown. β-actin was used as a loading control. Mean density of each protein was quantified from various blots by ImageJ software, and the values were plotted in terms of relative protein expression. (C) RAW macrophages (I) , CD11b+ peritoneal (II) , and bone marrow–derived macrophages (III) were treated with S-1P for indicated time intervals. Cell culture supernatants were collected, and IFN-γ (C) and IL-6 (D) were measured by ELISA at 24 and 48 h, respectively. Shown here is the pg/ml of cytokines released during the course of infection ± SEM from three independent experiments. Statistical analyses were conducted using two-way ANOVA followed by Bonferroni posttest (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: IFN-γ and IL-6 ELISA kits were purchased from R&D Systems (Darmstadt, Germany).

Techniques: Expressing, Control, Software, Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Infection

Journal: Frontiers in Immunology

Article Title: Sphingosine-1-Phosphate (S-1P) Promotes Differentiation of Naive Macrophages and Enhances Protective Immunity Against Mycobacterium tuberculosis

doi: 10.3389/fimmu.2019.03085

Figure Lengend Snippet: Lung homogenates and serum were prepared from S-1P conditioned mice and IFN-γ ( A ; both pulmonary compartment and serum) and IL-6 ( B ; both pulmonary compartment and serum) titres were quantified by ELISA. Similarly, IFN-γ and IL-6 titres were quantified from lungs homogenates of mice treated with FTY720 (C) and DHS (D) by ELISA. Shown here is the pg/ml of cytokines produced ± SEM from several mice used in each group. Statistical analysis were conducted using two-way Anova followed by Bonferroni post-test (** P < 0.01, *** P < 0.001).

Article Snippet: IFN-γ and IL-6 ELISA kits were purchased from R&D Systems (Darmstadt, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Produced

Journal: Frontiers in Immunology

Article Title: Pathological neutrophil extracellular traps hinder postoperative anal fistula wound healing and are attenuated by Zuoqing granule via suppression of the Nox4 pathway

doi: 10.3389/fimmu.2025.1730184

Figure Lengend Snippet: ZQG attenuates PMA-induced NETosis and inflammatory responses in human neutrophils via targeting the Nox4/ROS pathway. (A) ZQG suppresses oxidative burst and NET formation. Representative immunofluorescence images showing intracellular ROS (stained by DHE, red) and NETosis (marked by CitH3, green). Nuclei are counterstained with DAPI (blue). Scale bars: 20 μm. (B) Quantitative analysis of intracellular ROS levels. (C) Quantification of NETosis, expressed as the percentage of Sytox Green + cells. (D) Western blot analysis demonstrating the effects of ZQG and si-Nox4 on the protein expression of Nox4, p-Akt, and PADI4. (E) Densitometric quantification of Western blot results. (F) qPCR analysis of Nox4, PI3K, Akt, and PADI4 mRNA expression. (G) ELISA analysis of pro-inflammatory cytokine levels (IL-2, IL-5, IL-6, IL-12, TNF-α) in cell culture supernatants. Data are presented as mean ± SEM (n=10 independent experiments). ***p < 0.001 vs. PMA group.

Article Snippet: The concentrations of interleukin-2 (IL-2), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-12 (IL-12), and tumor necrosis factor-alpha (TNF-α) in the supernatants were quantified using specific commercial rat ELISA kits (R&D Systems, Catalog # R6000B) according to the manufacturers’ instructions.

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture