3150020b  (fluidigm)

Journal: Virologica Sinica

Article Title: Host factor Naf1 restricts HIV-1 infection of myeloid cells and compromises the capacity of dendritic cell to prime CD4 + T cell

doi: 10.1016/j.virs.2025.03.007

Figure Lengend Snippet: Naf1 knockdown increases NF-κB activation and cytokines expression. A Naf1 knockdown in MDDCs during the treatment with pseudo-typed HIV-luc/JRFL (10 ​ng p24 gag amount virus). B Expressions of cytokines and chemokines. MDDCs (5 ​× ​10 5 ​cells) with or without Naf1 knockdown were incubated in presence or absence of LPS (10 ​ng/mL) for 6 ​h, and cell total mRNAs were extracted, and the cytokines and chemokines were measured with real-time (RT-) PCR. Data are presented as mean ​± ​SD from three representative experiments. ∗∗∗ P ​< ​0.001 was considered as significant difference in unpaired t -test. C, D MDDCs (5 ​× ​10 5 ​cells) with or without Naf1 knockdown were used to detect the surface expressions of CD83, CD86 and HLA-DR with flow cytometry, and results from three donors were summarized ( D ).

Article Snippet: Monoclonal anti-human antibodies against the following molecules were used in immunostaining: PE-CD83 (HB15e, eBioscience, San Diego, CA, USA); PE-CD86 (IT2.2, eBioscience); APC-cy7-HLA-DR (LN3, eBioscience); PE-ICAM-1 (HA58, eBioscience); PerCP-cy5.5-CD3 (OKT3, eBioscience) and PE-CD69 (FN50, eBioscience).

Techniques: Knockdown, Activation Assay, Expressing, Virus, Incubation, Quantitative RT-PCR, Flow Cytometry

Journal: Frontiers in Nutrition

Article Title: HMOS 2’FL and 3FL prevent house dust mite induced proinflammatory cytokine release in vitro and decrease specific IgE production in a murine allergic asthma model

doi: 10.3389/fnut.2025.1491430

Figure Lengend Snippet: Mucosal immune response of BEC/DC/T cell upon HDM exposure. (A) A schematic overview of the coculture steps in this human in vitro bronchial mucosal immune model (adapted from ). Calu-3 bronchial epithelial cells (BEC) were cultured for 14 days in air-liquid interface (ALI) prior to coculture with monocyte-derived dendritic cells (moDCs). BEC-DCs and DCs alone were apically exposed to 10 μg/ml HDM for 24 h. After 24 h of exposure to HDM, primed DCs were collected for analysis and coculture with allogenic naïve Th cells for 5 days. Upon 24 h of HDM exposure, supernatants and moDCs were collected to measure secreted levels of (B) IL33, (C) TSLP, (D) TGFβ, (E) IL8 and determine the percentage of moDCs expressing the costimulatory markers (F) CD80 and (G) CD86. After subsequent coculture with of primed DCs with naïve Th cells, supernatants and cells were collected to measure the percentage of (H) CRTH2 and (I) IL13 expressing cells and secreted (J) IL4 as part of the T helper 2 cell response. In addition, the T helper 1 response was analyzed based on the percentage of (K) CXCR3 and (L) IFNγ expressing cells and secreted (M) IFNγ. The regulatory T cell response was determined by identification of the percentage of dual expressing (N) FoxP3 and CD25 cells and secretion of (O) IL10. Finally, (P) the ratio of type 2 IL4 and type 1 IFNγ secretion was calculated. Data is analyzed by paired t -tests, n = 6 biologically different donors (2 independent experiments each using 3 different donors), mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001). Some parts of this figure were created with BioRender.com

Article Snippet: Titrated amounts of the following antibodies were used to stain in vitro samples: CD11c-PerCP (3.9), HLA-DR-PE (LN3), CD80-FITC (2D10.4), CD86-PE/Cy7 (IT2.2) (All from eBioscience, USA), CD4-PerCP (OKTO4, eBioscience), CXCR3-Alexa Fluor 488 (1C6/CXCR3, BD Biosciences), CRTH2-APC (BM16, BD Biosciences), FoxP3-eFluor 660 (PCH101, Invitrogen), CD25-Alexa Fluor 488 (BC96, eBioscience), and IL13-PE (85BRD, eBioscience), and IFNγ-Amcyan (4S.B3, Biolegend).

Techniques: In Vitro, Cell Culture, Derivative Assay, Expressing

Journal: Frontiers in Nutrition

Article Title: HMOS 2’FL and 3FL prevent house dust mite induced proinflammatory cytokine release in vitro and decrease specific IgE production in a murine allergic asthma model

doi: 10.3389/fnut.2025.1491430

Figure Lengend Snippet: The immunomodulatory effects of HMOS were tested in this human in vitro bronchial mucosal immune model by preincubation of HMOS prior to HDM exposure. (A) Calu-3 BECs were cultured in ALI for 14 days before coculture with moDCs and basolateral preincubation with 0,01-0,05% 2’FL or 3FL for 24 h. Next, BEC/DC were apically exposed to 10 μg/ml HDM for 24 h. Followed by a coculture of primed moDCs with naïve Th cells for 5 days. After HMOS preincubation and HDM exposure, primed DCs were collected for analysis and coculture with allogenic naïve Th cells for 5 days (adapted from . Upon HMOS preincubation and HDM exposure, supernatants and moDCs were collected to measure secreted levels of (B) IL33, (C) TSLP, (D) TGFβ, (E) IL8 and determine the percentage of moDCs expressing the costimulatory markers (F) CD80 and (G) CD86. After subsequent coculture with of primed DCs with naïve Th cells, supernatants and cells were collected to measure the percentage of (H) CRTH2 and (I) IL13 expressing cells and secreted (J) IL4 as part of the T helper 2 cell response. In addition, the T helper 1 response was analyzed based on the percentage of (K) CXCR3 and (L) IFNγ expressing cells and secreted (M) IFNγ. The regulatory T cell response was determined by identification of the percentage of dual expressing (N) FoxP3 and CD25 cells and secretion of (O) IL10. Finally, (P) the ratio of type 2 IL4 and type 1 IFNγ secretion was calculated. When data fit a normal distribution and had an equal variability of differences, analysis was performed by One-Way ANOVA followed by a Dunnett’s multiple comparisons test. When these criteria were not met, data was transformed, a Geisser–Greenhouse correction was performed or a Friedman-test with Dunn’s multiple comparisons test was performed, n = 6 biologically different donors (2 independent experiments each using 3 different donors, mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001). Controls of are the same as those depicted in . Some parts of this figure were created with BioRender.com

Article Snippet: Titrated amounts of the following antibodies were used to stain in vitro samples: CD11c-PerCP (3.9), HLA-DR-PE (LN3), CD80-FITC (2D10.4), CD86-PE/Cy7 (IT2.2) (All from eBioscience, USA), CD4-PerCP (OKTO4, eBioscience), CXCR3-Alexa Fluor 488 (1C6/CXCR3, BD Biosciences), CRTH2-APC (BM16, BD Biosciences), FoxP3-eFluor 660 (PCH101, Invitrogen), CD25-Alexa Fluor 488 (BC96, eBioscience), and IL13-PE (85BRD, eBioscience), and IFNγ-Amcyan (4S.B3, Biolegend).

Techniques: In Vitro, Cell Culture, Expressing, Transformation Assay