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: BMC immunology

Article Title: TRPV1 + neurons alter Staphylococcus aureus skin infection outcomes by affecting macrophage polarization and neutrophil recruitment.

doi: 10.1186/s12865-023-00584-x

Figure Lengend Snippet: Fig. 3 TRPV1+ neurons affect the polarization of local macrophages in the skin. a IHC staining of the skin of TRPV1−/− and WT mice; scale bars, 500 μm; high magnification images, scale bars, 50 μm. b-d IHC analysis of the ratio of M0 (F4/80 +), M1 (CD80 +) and M2 (CD206 +) cells in TRPV1−/− and WT mice (n = 5/group); unpaired t test. e–h Flow cytometric analysis of M0 (CD11b + F4/80 +), M1 (CD11b + F4/80 + CD80 +), and M2 (CD11b + F4/80 + CD206 +) ratios in the skin of TRPV1−/− and WT mice (n = 5/group); unpaired t test. i-k Tissue expression levels of TNFα, IL-1β and IL-10 in TRPV1−/− and WT mice 1 day after skin infection; the control group was uninfected WT mice; One-way ANOVA with Tukey’s post hoc test. Data were pooled from two or three independent experiments

Article Snippet: After washing, the cells were fixed/permeabilized according to the instructions provided by the manufacturer (MultiSciences, FoxP3/Transcription Factor Staining Buffer Kit) and then incubated on ice with CD206 (Elabscience, #E-AB-F0992C) for 30 min. Flow cytometry data were collected and exported using Beckman CytoFLEX (USA).

Techniques: Immunohistochemistry, Expressing, Infection, Control

Journal: BMC immunology

Article Title: TRPV1 + neurons alter Staphylococcus aureus skin infection outcomes by affecting macrophage polarization and neutrophil recruitment.

doi: 10.1186/s12865-023-00584-x

Figure Lengend Snippet: Fig. 5 CGRP regulates the polarization of BMDMs and the release of inflammatory factors. a-c BMDMs cultured in vitro were induced to M1 polarization with IFN-γ and M2 polarization with IL-4 The polarized macrophages were treated with CGRP or PBS and stained with DAPI (blue), CD80 (green), and CD206 (red); the ratio of CD80 + and CD206 + was analyzed under different intervention conditions. No difference in the confluency of BMDMs was observed among different groups. Scale bars, 20 μm. One-way ANOVA with Tukey’s post hoc test. d-f Expression levels of TNFα, IL-1β, and IL-10 in BMDMs after polarization in vitro under different experimental conditions; one-way ANOVA with Tukey posttests. Data were pooled from two or three independent experiments

Article Snippet: After washing, the cells were fixed/permeabilized according to the instructions provided by the manufacturer (MultiSciences, FoxP3/Transcription Factor Staining Buffer Kit) and then incubated on ice with CD206 (Elabscience, #E-AB-F0992C) for 30 min. Flow cytometry data were collected and exported using Beckman CytoFLEX (USA).

Techniques: Cell Culture, In Vitro, Staining, Expressing

Journal: Cells

Article Title: Cancer Immunomodulatory Effect of Bidens pilosa L. in Mice: Suppression of Tumor-Associated Macrophages and Regulatory T Cells

doi: 10.3390/cells15020126

Figure Lengend Snippet: Expression levels of mRNAs and proteins associated with TAMs and Tregs in BPA -treated mouse tumors. ( A ) Quantification of mRNA by qRT-PCR for TAM/Treg-associated tumor-promoting factors (Arg-1, YM-1, CCL2, CCL22, Foxp3, CD25, TGF-β, CXCR3, CCR4, CCR8, CCR10) and tumor-inhibitory factors (TNF-α, IL-1β, iNOS, CXCL10, CCR7). Data were normalized to 18 S rRNA and are shown relative to the model group (baseline control). ( B ) Representative immunoblots of tumor tissue for TAM- and Treg-associated proteins. Pro-tumor: CD206, Foxp3, CD25, PD-1, PD-L1; Anti-tumor: CD80, CD4, GITR. ( C ) Quantification of protein expression from Western blots. Values were normalized to α-tubulin, β-actin, or GAPDH as indicated. Statistical significance is denoted as follows ( n = 6): compared to the model group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To block Fc receptors, cells were incubated with anti-CD16/32 (Elabscience, Cat. E-AB-F0997A, Wuhan, China) for 10 min. After washing, cells were stained with PerCP/Cyanine 5.5 anti-mouse F4/80 (Elabscience, Cat. E-AB-F0995J), FITC anti-mouse CD206 antibody (Elabscience, Cat. E-AB-F1135C), and APC anti-mouse CD80 antibody (Elabscience, Cat. E-AB-F0992E) at 4 °C for 30 min.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot

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