Journal: ACS Biomaterials Science & Engineering

Article Title: Methods and Technical Issues for Optimizing the Production of Hydrogels Containing Decellularized Wharton’s Jelly

doi: 10.1021/acsbiomaterials.5c02006

Figure Lengend Snippet: Human M1-polarized macrophages were exposed to A075G3 and A075W3 for 48 h. Positivity for CD80, a pro-inflammatory surface marker, was assessed by flow cytometry and expressed as mean fluorescence intensity (MFI) ± SD ( n = 4 for CTR, A075G3 and A075W3). * p < 0.05 (A075W3 vs CTR).

Article Snippet: PE conjugated antihuman CD80 Antibody (clone REA661) from Miltenyi Biotec (Germany).

Techniques: Marker, Flow Cytometry, Fluorescence

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: FIGURE 1 | 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: LC-3, CD80, CD86, and MHC-II 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 Frontiers in Pharmacology | www.frontiersin.org March 2022 | Volume 13 | Article 8377843 Frontiers in Pharmacology | www.frontiersin.org March 2022 | Volume 13 | Article 8377844 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, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Staining, 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: FIGURE 11 | 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: LC-3, CD80, CD86, and MHC-II 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 Frontiers in Pharmacology | www.frontiersin.org March 2022 | Volume 13 | Article 8377843 Frontiers in Pharmacology | www.frontiersin.org March 2022 | Volume 13 | Article 8377844 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, Staining, Cytometry

Journal:

Article Title: Toxoplasma gondii regulates recruitment and migration of human dendritic cells via different soluble secreted factors

doi: 10.1111/j.1365-2249.2005.02856.x

Figure Lengend Snippet: Phenotype of DC after 7 days of culture in vitro before contact with T. gondii

Article Snippet: Cells (1·5 × 10 5 ) were incubated with 10 μl labelled monoclonal antibodies (MoAb) for 30 min at 4°C: CD1a (IgG2a; Dako, Trappes, France), CD80-FITC (IgG2b k; Diaclone, Besancon, France), CD83-FITC (IgG2 k; Immunotech, Marseille, France), CD86-FITC (IgG1), CD40 (IgG1; Diaclone); CCR6 (IgM; BD Bioscience, Pont de Claix, France), CCR7 (IgG2B; R & D Systems, Minneapolis, MN, USA) and HLA-DR (IgG2a; Beckton Dickinson, Mountain View, CA, USA).

Techniques: In Vitro

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: Fig. 3 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 per formed 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: Fig. 4 HUCMSC-migrasomes impaired DC maturation and function in vitro. A BMDCs incubated with Dil-labeled migrasomes under fluorescence micro scope (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 × 104) from WT mice were pre-treated with or without migrasomes and then pulsed with OVA323–339 and LPS and incubated at a 1:5 ratio with splenic CD4+ T cells isolated from OT-II mice (1 × 105) 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, Isolation, Cell Culture, Expressing, Real-time Polymerase Chain Reaction

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: In vitro study of cold atmospheric plasma-activated liquids inhibits malignant melanoma by affecting macrophage polarization through the ROS/JAK2/STAT1 pathway.

doi: 10.1016/j.biopha.2024.116657

Figure Lengend Snippet: Fig. 4. PAL treatment affects M0 and M2 type RAW264.7 macrophage differentiation. (A) Percentages of different cell subpopulations of M0 and M2 type RAW264.7 cells with or without PAL treatment were determined by flow cytometry and a histogram illustrating the percentage of M0 and M2 type macrophages of RAW264.7 cells was generated; (B) qPCR was used to analyze the expression levels of CD80 and CD206-related genes relative to GAPDH (a housekeeping gene) in M0 and M2 type RAW264.7 cells and THP1 cells with or without PAL treatment. The data are expressed as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: An antiPerCP/Cy5.5-conjugated anti-CD80 antibody (Elabscience, Wuhan, CN, Cat. No. E-AB-F0992J) and a PE-conjugated anti-CD206 antibody (BioLegend, CA, USA, Cat. No. 141705) were incubated with RAW264.7 cells at room temperature for 30 minutes.

Techniques: Flow Cytometry, Generated, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: In vitro study of cold atmospheric plasma-activated liquids inhibits malignant melanoma by affecting macrophage polarization through the ROS/JAK2/STAT1 pathway.

doi: 10.1016/j.biopha.2024.116657

Figure Lengend Snippet: Fig. 7. PAL treatment promotes the differentiation of unactivated M0 type macrophages and tumor-promoting M2 type macrophages to tumor-resistant M1 type macrophages via the ROS/JAK2/STAT1 pathway. (A) Immunoblotting was performed using anti-CD206 and anti-CD80 antibodies to observe the protein expression of CD80 and CD206 after treating M0 type RAW264.7 and THP1 cells under various conditions. GAPDH protein was used as a loading control; (B) immunoblotting was performed using anti-CD206 and anti-CD80 antibodies to observe M2 type RAW264.7 cells under different conditions, the expression of CD80 and CD206 after THP1 cells was analyzed. GAPDH protein was used as a loading control; (C) the expression of M1 type macrophage-specific markers TNF-α, IL-6, and iNOS-related genes relative to GAPDH (housekeeping gene) was analyzed by qPCR after PAL treatment of M0 type RAW264.7 and THP1 cells; (D) the expression of M1 type macrophage-specific markers TNF-α, IL-6, and iNOS, and M2 type macrophage-specific markers Arg1, MRC1, and Chi3l3-related genes were analyzed by qPCR after PAL treatment of M2 type RAW264.7 and THP1 cells relative to the GAPDH (a housekeeping gene); (E, F) concentrations of the M1 type macrophage-specific markers TNF-α, IL-6, IL-1β, and iNOS in the culture broth after PAL treatment of M0 type RAW264.7 cells and THP1 cells were measured using an enzyme-linked immu nosorbent assay (ELISA) kit; (G, H) concentrations of the M1 type macrophage-specific markers TNF-α, IL-6, IL-1β, and iNOS, and the M2 type macrophage-specific markers Arg1, MRC1, and Chi3l3 in the culture broth after PAL treatment of M2 type RAW264.7 cells and THP1 cells were detected using an enzyme-linked immunosorbent assay (ELISA) kit. G1 served as the negative control group; G2 as the PAL-treated group; G3 as the PAL + CAT-treated group; and G4 as the PAL + F-ara-A-treated group. The data are expressed as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. CAT: catalase; F-ara-A: Fludarabine.

Article Snippet: An antiPerCP/Cy5.5-conjugated anti-CD80 antibody (Elabscience, Wuhan, CN, Cat. No. E-AB-F0992J) and a PE-conjugated anti-CD206 antibody (BioLegend, CA, USA, Cat. No. 141705) were incubated with RAW264.7 cells at room temperature for 30 minutes.

Techniques: Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Characterization of immune cell subtypes in three commonly used mouse strains reveals gender and strain-specific variations

doi: 10.1038/s41374-018-0137-1

Figure Lengend Snippet: Details of antibodies used in the study

Article Snippet: CD80 , APC , Miltenyi , 16–10A1 , 130–102-584 , 3.75 pL.

Techniques: Concentration Assay