Journal: Arthritis Research & Therapy

Article Title: Interferon-induced protein IFIT4 is associated with systemic lupus erythematosus and promotes differentiation of monocytes into dendritic cell-like cells

doi: 10.1186/ar2475

Figure Lengend Snippet: Comparison of the phenotypic profiles of DCLCs primed with or without IFIT4 over-expression. (a) Surface antigens of normal THP-1 cells were examined by flow cytometry. (b) To analyze the effect of IFIT4 on phenotypic changes of dendritic cell-like cells (DCLCs) during the process of differentiation, THP-1 cells were transfected with pEGFP-IFIT4 or pEGFP-C1; 36 hours later, cells were stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF; 50 ng/ml) and IL-4 (20 ng/ml) for 90 hours to generate DCLCs. These DCLCs primed with pEGFP-IFIT4 or pEGFP-C1 transfection were incubated with fluorochrome-conjugated monoclonal antibodies (mAbs) and the antigens of CD40, CD80, CD86, CD83, HLA-DR, CD14 and CD1a on the surface of those DCLCs were analyzed by flow cytometry. Appropriate fluorochrome or isotype control mAbs of each antibody species were used as negative controls. Shaded histograms represent isotype control antibodies. The thick line represents DCLCs primed with pEGFP-IFIT4 transfection, whereas the slender lines represent DCLCs primed with pEGFP-C1 transfection. All experiments were performed three times and a set of representative histograms was presented. IFIT4, interferon-induced protein with tetratricopeptide repeats 4.

Article Snippet: FITC anti-human CD40 (Catalogue no: FAB617F) and PE anti-human CD80 (Catalogue no: FAB140P) were from R&D Systems, Minneapolis, MN, USA.

Techniques: Comparison, Over Expression, Flow Cytometry, Transfection, Incubation, Bioprocessing, Control

Journal: Frontiers in Pharmacology

Article Title: Chlorin e6-Induced Photodynamic Effect Polarizes the Macrophage Into an M1 Phenotype Through Oxidative DNA Damage and Activation of STING

doi: 10.3389/fphar.2022.837784

Figure Lengend Snippet: Ce6 PDT remodeled macrophages into the M1 phenotype in vitro . Macrophages were treated by Ce6 PDT (Ce6 was loaded at a concentration of 4 μg/ml, the loading time was 12 h, the irradiation time was 20/40/60 s, and the placement time was 8 h). (A,B) Phagocytic ability of macrophages was assayed through the fluorescence latex beats experiment and confocal microscopy. The average number of latex beats engulfed by macrophages was counted. (C–E) Expressions of GBP5 and iNOS were measured by WB. The blots were quantitatively analyzed using the ratio of mean gray. (F,G) mRNA levels of IL-1β and IL-6 were quantified through RT-PCR. (H–M) Surface CD80, CD86, and MHC-II expressions were detected by immunofluorescence staining and flow cytometry. Geometric means were used to quantify the MFI. (N,O) LLC were co-cultured with macrophages treated with Ce6, as described before. The apoptosis rate of LLC was detected by Annexin-V/PI double staining and flow cytometry. Values were means ± SD ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: For the detection of NF-κB, LC-3, and MHC-II expressions or location, cells were incubated with primary antibodies of NF-κB (10745-1-AP, Proteintech, Wuhan, China), LC-3 (12741S, CST, Boston, United States), CD80 (E-AB-F0992D, Elabscience), CD86 (E-AB-F0994D, Elabscience), and MHC-II (sc-66205, Santa Cruz Biotechnology, Santa Cruz, United States) overnight at 4°C and then incubated with the goat anti-rat IgG/Alexa Fluor 488 secondary antibody (bs-0293G-AF488, Bioss, Beijing, China) for another 60 min and washed three times before confocal microscopy (FV3000RS, Olympus, Japan) or flow cytometry (CytoFLEX, Beckman Coulter, United States).

Techniques: In Vitro, Concentration Assay, Irradiation, Fluorescence, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Flow Cytometry, Cell Culture, Double Staining

Journal: Frontiers in Pharmacology

Article Title: Chlorin e6-Induced Photodynamic Effect Polarizes the Macrophage Into an M1 Phenotype Through Oxidative DNA Damage and Activation of STING

doi: 10.3389/fphar.2022.837784

Figure Lengend Snippet: Suppression of the STING molecule attenuated the Ce6 PDT-induced M1 phenotype remodeling of macrophages. The STING molecule in macrophages was silenced by SiRNA (100 pM) for 8 h and then treated with Ce6, as described before. (A–C) The expression of cGAS and p-NF-κB in macrophages was measured by WB. The blots were quantitatively analyzed using the ratio of mean gray. (D–F) The molecules of iNOS and GBP5 were analyzed by WB. The blots were quantitatively analyzed using the ratio of mean gray. (G,H) The expression of the STING was detected by WB. The blots were quantitatively analyzed using the ratio of mean gray. (I–N) The surface expression of MHC-II, CD80, and CD86 was analyzed by immunofluorescence staining and flow cytometry. Geometric means were used to quantify the MFI. Values were means ± SD ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: For the detection of NF-κB, LC-3, and MHC-II expressions or location, cells were incubated with primary antibodies of NF-κB (10745-1-AP, Proteintech, Wuhan, China), LC-3 (12741S, CST, Boston, United States), CD80 (E-AB-F0992D, Elabscience), CD86 (E-AB-F0994D, Elabscience), and MHC-II (sc-66205, Santa Cruz Biotechnology, Santa Cruz, United States) overnight at 4°C and then incubated with the goat anti-rat IgG/Alexa Fluor 488 secondary antibody (bs-0293G-AF488, Bioss, Beijing, China) for another 60 min and washed three times before confocal microscopy (FV3000RS, Olympus, Japan) or flow cytometry (CytoFLEX, Beckman Coulter, United States).

Techniques: Expressing, Immunofluorescence, Staining, Flow Cytometry

Journal: Stem Cell Research & Therapy

Article Title: Migrasomes derived from human umbilical cord mesenchymal stem cells: a new therapeutic agent for ovalbumin-induced asthma in mice

doi: 10.1186/s13287-025-04145-4

Figure Lengend Snippet: Administration of hUCMSC-migrasomes diminished Th2 response in allergic asthma by suppressing DC maturation. A The expression of IL-4, IL-5 and IL-13 in the lung of mice in each group were detected by real-time PCR ( n = 6). B Gating strategy and representative flow cytometric images for the analysis of CD45 + CD4 + CCR4 + CXCR3 − Th2 cells in the lungs of mice from each group. C Graphs showing the % of CD45 + CD4 + CCR4 + CXCR3 − Th2 cells in the lungs of mice from each group ( n = 6). D Gating strategy and representative flow cytometric dot plots (left) for the analysis of CD11b + DCs in the lungs of allergic mice and the numbers of CD11b + DCs (right) in the lungs of mice from each group ( n = 6). E The mean MFI of CD80, CD86 and MHC-II on CD11b + DCs ( n = 4–6). F The expression of IL-6 in the lung of mice were detected by real-time PCR ( n = 6). G Distribution of Dir-labeled migrasomes in OVA-induced mice lung after tail vein administration (17-day injection and 18-day detected). H Representative immunofluorescence images of Dil-labeled migrasomes (red) taken up by CD11c + cells or CD4 + cells. Data were presented as mean ± SD. A one-way analysis of variance (Tukey Kramer post hoc tests) was performed on the data. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The cell pellets were then stained with a range of monoclonal antibodies including FITC anti-mouse CD45 (eBioscience, San Diego, CA), eflour 450 anti-mouse CD4 (eBioscience), PE- anti-mouse CCR4 (Biolegend), APC anti-mouse CXCR3 (Biolegend), eflour 450 anti-mouse CD11c (eBioscience), FITC anti-mouse CD11b (Elabscience, Wuhan, China), PE/Cyanine7 anti-mouse CD80 (Elabscience), PE anti-mouse CD86 (eBioscience) and APC anti-mouse MHC-II (eBioscience).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Labeling, Injection, Immunofluorescence

Journal: Stem Cell Research & Therapy

Article Title: Migrasomes derived from human umbilical cord mesenchymal stem cells: a new therapeutic agent for ovalbumin-induced asthma in mice

doi: 10.1186/s13287-025-04145-4

Figure Lengend Snippet: HUCMSC-migrasomes impaired DC maturation and function in vitro. A BMDCs incubated with Dil-labeled migrasomes under fluorescence microscope (white bar = 25 μm). B The cell viability of BMDCs treated by hUCMSC-migrasomes for 24 h (left) and 48 h (right) ( n = 4). C BMDCs were isolated from wild-type BALB/c mice and cultured at a density of 2 × 10⁵ cells/mL. The cells were stimulated with OVA (100 µg/mL) and LPS (10 ng/mL) for 48 h, with or without hUCMSC-migrasomes (20 µg/mL) pre-treated. After that, cells were collected for the detection of DC maturation markers including CD80, CD86 and MHC-II ( n = 3). The representative images for the detection of DC maturation markers are displayed. D Statistical analysis of the mean MFIs of CD80, CD86 and MHC-II of BMDCs ( n = 3). E Statistical analysis of the expression of IL-6 in BMDCs by real-time PCR ( n = 3). F BMDCs (2 × 10 4 ) from WT mice were pre-treated with or without migrasomes and then pulsed with OVA 323–339 and LPS and incubated at a 1:5 ratio with splenic CD4 + T cells isolated from OT-II mice (1 × 10 5 ) for 72 h. G Statistical analysis of the expression of IL-4, IL-5, IL-13 in collected cell by real-time PCR ( n = 3). Data were presented as mean ± SD. A one-way analysis of variance (Tukey Kramer post hoc tests) was performed on the data. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The cell pellets were then stained with a range of monoclonal antibodies including FITC anti-mouse CD45 (eBioscience, San Diego, CA), eflour 450 anti-mouse CD4 (eBioscience), PE- anti-mouse CCR4 (Biolegend), APC anti-mouse CXCR3 (Biolegend), eflour 450 anti-mouse CD11c (eBioscience), FITC anti-mouse CD11b (Elabscience, Wuhan, China), PE/Cyanine7 anti-mouse CD80 (Elabscience), PE anti-mouse CD86 (eBioscience) and APC anti-mouse MHC-II (eBioscience).

Techniques: In Vitro, Incubation, Labeling, Fluorescence, Microscopy, Isolation, Cell Culture, Expressing, Real-time Polymerase Chain Reaction

Journal:

Article Title: Lack of Tumor Necrosis Factor Alpha Induces Impaired Proliferation of Hepatitis B Virus-Specific Cytotoxic T Lymphocytes

doi: 10.1128/JVI.77.4.2469-2476.2003

Figure Lengend Snippet: Comparison of DC function in TNF-α+/+ and TNF-α−/− mice. CD11c+ cell separation was carried out by using the VarioMACS system. (A) Flow cytometric analysis of the expression of the MHC-I molecule H-2Ld (restriction element for HBsAg-specific CTL), the MHC-II molecule I-A/I-E, CD80, and CD86 in TNF-α+/+ and TNF-α−/− DCs. All samples were assayed in duplicate. (B) The allostimulatory capacity of CD11c+ DCs from TNF-α+/+ and TNF-α−/− mice was assessed by the MLR as described in Materials and Methods with allogeneic murine CD4+ T cells from AKR mice (H-2k). Each data point and error bar represent the mean and SD, respectively, of results for triplicate samples.

Article Snippet: To evaluate the expression intensities of MHC and costimulatory molecules, mouse or rat biotin-conjugated anti-mouse MHC-I ( H - 2L d ), MHC-II ( I - A d / I - E d ), PE-labeled streptavidin (BD PharMingen), rat PE-conjugated anti-mouse CD80 and CD86 (Cedarlane, Ontario, Canada) were used for staining.

Techniques: Expressing