cd86 monoclonal antibody (it2.2)-pe  (Thermo Fisher)

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

Journal: bioRxiv

Article Title: CD300e as a Driver of Immunosuppressive Tumor Microenvironment in Colorectal Cancer

doi: 10.1101/2024.09.01.610700

Figure Lengend Snippet: A ) CD300e total content was evaluated by Western blot in matched NM and CRC tissue. Blot refers to three representative CRC patients (P1, P2, and P3). Quantification of CD300e was performed by densitometry and normalized to Vinculin as housekeeping protein. Data are expressed as n -fold of matched CRC vs NM tissue (mean ± SD of 8 CRC patients). B-D ) mRNA expression of CD300e (B), MHC-II ( C ) and CIITA ( D ) was evaluated in matched NM and CRC tissue by qRT-PCR. Data were normalized to β-actin as an endogenous reference gene and are expressed as 2 -ΔΔCt of CRC tissue relative to matched NM tissue (mean ± SD of 6 CRC patients). E ) Representative images from NM and CRC samples sequentially immunostained for MHC-II (red) and CD163 (blue)/CD300e (brown). Arrows highlight CD300e + /MHC-II low macrophages. The histogram shows the percentage of CD300e + /MHC-II low macrophages in NM and CRC tissue expressed as mean ± SD of 8 CRC patients. F ) Scheme of normal colon organoid (NCO) and tumor colon organoid (TCO) generation from CRC patients’ tissue (upper panel) and of the co-culture between NCO or TCO with monocytes derived from healthy donors (lower panel). The image has been created with Biorender. G ) Surface expression and mRNA expression of CD300e were evaluated by flow cytometry and qRT-PCR, respectively, on monocyte-derived macrophages after 5 d of co-culture with either NCO or TCO. Surface expression is expressed as n-fold vs macrophages co-cultured with NCO ± SD. mRNA expression was normalized on β-actin as an endogenous reference gene and is expressed as 2 -ΔΔCt relative to monocyte-derived macrophages cultured with NCO, ± SD (n = 4 different monocyte donors co-cultured with 4 NCO and 4 TCO lines). H ) The surface expression of MHC-II, CD86, and CD206 was evaluated by flow cytometry on monocyte-derived macrophages after 5 d of co-culture with either NCO or TCO. Data are expressed as n-fold vs macrophages co-cultured with NCO ± SD of the experiments in G ). I ) mRNA expression of CD163, CD206, IL-10, IL-6, and TGF-β was evaluated on macrophages after 5 d of co-culture as in H ). Data were normalized on β-actin as an endogenous reference gene and expressed as 2 -ΔΔCt relative to macrophages co-cultured with NCO, ± SD of the experiments in G ). J ) Release of IL-6 and IL-10 in the co-culture supernatants collected after 5 d of co-culture of macrophages with either NCO or TCO by ELISA assay. Data are expressed as n-fold vs supernatants of macrophages co-cultured with NCO ± SD of the experiments in G ). Statistical significance was determined by Student’s t-test. CRC = colorectal cancer; NM = normal colonic mucosa; NCO = normal colon organoids; TCO = tumor colon organoids. *=p≤0.05; **= p≤0.01; ***=p≤0.001; ****=p≤0.0001.

Article Snippet: Cells were stained with the following antibodies: APC anti-MHC-II (clone L243, eBiosciences, San Diego, CA, USA), BB515 anti-CD206 (clone 19.2, eBiosciences), PE-Cy7 anti-CD86 (clone IT2.2, eBiosciences) and PE anti-CD300e (clone UP-H2, Invitrogen).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Co-Culture Assay, Derivative Assay, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: CD300e as a Driver of Immunosuppressive Tumor Microenvironment in Colorectal Cancer

doi: 10.1101/2024.09.01.610700

Figure Lengend Snippet: A ) mRNA expression of CD300e and MHC-II was evaluated by qRT-PCR in murine macrophages before (t0) and after 48 hours of co-culture with tumor colon organoids (+ TCO). Data were normalized on β-actin and 18S and as endogenous reference genes and are expressed as 2 -ΔΔCt relative to macrophages at t0 (mean ± SD, n = 6 CD300e +/+ macrophage donors co-cultured with 3 different TCO lines). B) The mRNA expression of macrophage immunostimulatory (CD38, CD86, iNOS, MHC-II, TNF-α) and immunosuppressive (EGR2, CD163, CD206, Arginase1, TGF-β) markers was evaluated by qRT-PCR on CD300e +/+ and CD300e -/- macrophages after 48 hours of co-culture with TCO. Data were normalized on β-actin and 18S and as endogenous reference genes and are expressed as 2 -ΔΔCt relative to CD300e +/+ macrophages (mean ± SD of 9 CD300e +/+ and 9 CD300e -/- macrophage donors, co-cultured with 3 different TCO lines). C ) After 48 hours of co-culture with TCO, macrophages were incubated for 30 minutes with Zymosan A BioParticles™ to evaluate their phagocytic ability. Representative confocal images of CD300e +/+ and CD300e -/- macrophages ± Cytocalasin D are shown. Nuclei are stained with DAPI (blue), F4/80 was used as macrophage marker (green) and Zymosan BioParticles™ are visualized in red. Scale bar = 50 μm. The number of internalized Zymosan A BioParticles™ was manually counted; data are expressed as ratio of the number of Zymosan A BioParticles on the number of phagocytic macrophages (macrophages that internalized ≥1 bioparticle) of 3 CD300e +/+ and 3 CD300e -/- macrophage donors exposed to 2 different TCO lines. Ten images per condition were analyzed. D,E ) After 48 hours of co-culture with TCO, macrophages were incubated for 2 hours with live MC38 cells ( D ) or 1 hour with apoptotic MC38 cells ( E ) to evaluate their capacity to phagocytose live (phagocytosis) or apoptotic (efferocytosis) tumor cells. Confocal images represent macrophages stained with Vybrant DiO (green) and MC38 cells stained with Vybrant DiD (red). Scale bar = 50 μm. The percentage of macrophages that phagocytosed MC38 cells was manually calculated on seven images per condition (n = 3 CD300e+/+ and 3 CD300e-/-macrophage donors). Statistical significance was determined by Student’s t-test. CytD = Cytocalasin D; NT = untreated; TCO = tumor colon organoids. *=p≤0.05; **= p≤0.01; ****=p≤0.0001.

Article Snippet: Cells were stained with the following antibodies: APC anti-MHC-II (clone L243, eBiosciences, San Diego, CA, USA), BB515 anti-CD206 (clone 19.2, eBiosciences), PE-Cy7 anti-CD86 (clone IT2.2, eBiosciences) and PE anti-CD300e (clone UP-H2, Invitrogen).

Techniques: Expressing, Quantitative RT-PCR, Co-Culture Assay, Cell Culture, Incubation, Staining, Marker