Journal: NPJ Vaccines

Article Title: A KIF20A-based thermosensitive hydrogel vaccine effectively potentiates immune checkpoint blockade therapy for hepatocellular carcinoma

doi: 10.1038/s41541-024-01060-2

Figure Lengend Snippet: a CD80 and CD86 levels of BMDCs in flow cytometry (The gating strategy was shown in the Supplementary Fig. ). b Quantification of the proportion of CD80 + CD86 + BMDCs. c MHC-I level of BMDCs indicated by flow cytometry (The gating strategy was shown in the Supplementary Fig. ). d Quantification of the proportion of MHC-I + BMDCs. e MHC-II level of BMDCs in flow cytometry (The gating strategy was shown in the Supplementary Fig. ). f Quantification of MHC-II + BMDCs. g CFDA-SE level in CD3 + T cells (The gating strategy was shown in the Supplementary Fig. ). h Quantification of T cell proliferation rate. i CLSM images of infiltrated CD8a + T cells into Hepa1-6 cell spheroids. j Quantification of T cell infiltration.

Article Snippet: Cells were subsequently stained with APC-conjugated anti-mouse CD11c, FITC-conjugated anti-mouse CD80, and PE-conjugated anti-mouse CD86 antibodies (Elabscience, China) for flow cytometry analysis (Beckman, USA).

Techniques: Flow Cytometry

Journal: NPJ Vaccines

Article Title: A KIF20A-based thermosensitive hydrogel vaccine effectively potentiates immune checkpoint blockade therapy for hepatocellular carcinoma

doi: 10.1038/s41541-024-01060-2

Figure Lengend Snippet: a Scheme illustration of in vivo treatment and monitoring (Created by the authors with Photoshop v22.5.6). b Bioluminescence images of tumor-bearing mice in different groups. c Relative signal intensity of tumors in different groups. d Body weight of the mice in different groups. e Flow cytometry analyzing CD80 and CD86 in CD11c + dendritic cells in mouse inguinal lymph nodes (Gating strategy was shown in the Supplementary Fig. ). f Quantification of the percentage of CD80 + CD86 + cells in CD11c + dendritic cells. g Flow cytometry analyzing CD8a and CD4 of T cells (CD45 + CD3 + ) in tumor tissues (Gating strategy was shown in the Supplementary Fig. ). h Quantification of the percentage of CD8a + cells in T cells.

Article Snippet: Cells were subsequently stained with APC-conjugated anti-mouse CD11c, FITC-conjugated anti-mouse CD80, and PE-conjugated anti-mouse CD86 antibodies (Elabscience, China) for flow cytometry analysis (Beckman, USA).

Techniques: In Vivo, Flow Cytometry

Journal: Journal of Inflammation (London, England)

Article Title: Tissue-plasminogen activator effects on the phenotype of splenic myeloid cells in acute inflammation

doi: 10.1186/s12950-024-00375-0

Figure Lengend Snippet: References of flow cytometry antibodies used for immunophenotyping

Article Snippet: CD80 , REA983 , APC-Vio770 , 130–116-463 , human IgG1/REA293 , Miltenyi Biotec.

Techniques: Flow Cytometry, Clone Assay

Journal: Journal of Inflammation (London, England)

Article Title: Tissue-plasminogen activator effects on the phenotype of splenic myeloid cells in acute inflammation

doi: 10.1186/s12950-024-00375-0

Figure Lengend Snippet: Effects of LPS treatment on the phenotype of splenic macrophages from tPA −/− mice. A Representative flow cytometry gating strategy used for quantification of spleen macrophages (F4/80 + CD11b + ), expressing MHCII molecules or not (F4/80 + CD11b + MHCII +or− ) and costimulatory molecules (F4/80 + CD11b + MHCII +or− CD80 + CD86 + ). B Frequency of macrophages (Sham WT n = 9; Sham tPA −/− ; LPS WT; LPS tPA −/− n = 10). C Frequency of MHCII − macrophages (F4/80 + CD11b + MHCII − ) and of costimulatory molecule expressing cells (F4/80 + CD11b + MHCII − CD80 + CD86 + ), (Sham WT n = 9; Sham tPA −/− ; LPS WT; LPS tPA −/− n = 10). D Frequency of MHCII + macrophages (F4/80 + CD11b + MHCII + ), (Sham WT n = 9; Sham tPA −/− ; LPS WT; LPS tPA −/− n = 10), MFI quantification of MHCII on MHCII + macrophages (Sham WT n = 9; Sham tPA −/− ; LPS WT; LPS tPA −/− n = 10) and frequency of MHCII + macrophages expressing costimulatory molecules (F4/80 + CD11b + MHCII + CD80 + CD86 + ), (Sham WT n = 9; Sham tPA −/− ; LPS WT; LPS tPA −/− n = 10). E MFI quantification of CD80 (Sham WT n = 8; Sham tPA −/− ; LPS WT n = 10; LPS tPA −/− n = 9), CD86 (Sham WT n = 8; Sham tPA −/− n = 9, LPS WT; LPS tPA −/− n = 10) and CD11b molecules (Sham WT n = 8; Sham tPA −/− ; LPS WT; LPS tPA −/− n = 10) on MHCII − macrophages. F MFI quantification of CD80 (Sham WT n = 8; Sham tPA −/− ; LPS WT n = 10; LPS tPA −/− n = 9), CD86 and CD11b molecules (Sham WT n = 9; Sham tPA −/− ; LPS WT; LPS tPA −/− n = 10) on MHCII + macrophages. Data are shown as individual animals with mean ± SD, two-way ANOVA with Bonferroni’s post-hoc

Article Snippet: CD80 , REA983 , APC-Vio770 , 130–116-463 , human IgG1/REA293 , Miltenyi Biotec.

Techniques: Flow Cytometry, Expressing

Journal: Journal of Inflammation (London, England)

Article Title: Tissue-plasminogen activator effects on the phenotype of splenic myeloid cells in acute inflammation

doi: 10.1186/s12950-024-00375-0

Figure Lengend Snippet: cDC phenotype was modulated in an inflammatory setting. A Representative flow cytometry gating strategy used for quantification of total DCs (F4/80 − CD11c + ) and cDCs: cDC1 (F4/80 − CD11c + CD11b − MHCII + ) and cDC2 (F4/80 − CD11c + CD11b + MHCII + ). B Quantification of total DC frequency (Sham WT n = 8; Sham tPA −/− ; LPS WT; LPS tPA −/− n = 10). C Frequency of cDC1, MFI of MHCII molecules on cDC1 and frequency of CD80 + CD86 + cDC1 (Sham WT n = 9; Sham tPA −/− ; LPS WT; LPS tPA −/− n = 10). D Frequency of cDC2 and CD80 + CD86 + cDC2, MFI of MHCII and CD11b on cDC2 (Sham WT n = 9; Sham tPA −/− ; LPS WT; LPS tPA −/− n = 10). Data are shown as individual animals with mean ± SD, two-way ANOVA with Bonferroni’s post-hoc

Article Snippet: CD80 , REA983 , APC-Vio770 , 130–116-463 , human IgG1/REA293 , Miltenyi Biotec.

Techniques: Flow Cytometry

Journal: STAR Protocols

Article Title: A protocol for high-throughput screening for immunomodulatory compounds using human primary cells

doi: 10.1016/j.xpro.2023.102405

Figure Lengend Snippet: Representative schematic of the high throughput flow cytometry system A total of 5 μL of antibodies diluted 1:200 in PBS were added to each well containing 30 μL of cells (50,00 cells/well). This flow cytometry system captures the total events or cells per well in addition to the antibody activity, which measures markers including the surface receptors Ox40, CD80/86, and HLA-DR. The flow cytometry approach also identifies B cells, T cells, and monocytes.

Article Snippet: CD80 Antibody, anti-human, PE, REAfinityTM (Clone- REA661). Diluted 1:200 with dPBS by volume. , Miltenyi Biotec , Order Number: 130-123-253.

Techniques: High Throughput Screening Assay, Flow Cytometry, Activity Assay

Journal: STAR Protocols

Article Title: A protocol for high-throughput screening for immunomodulatory compounds using human primary cells

doi: 10.1016/j.xpro.2023.102405

Figure Lengend Snippet:

Article Snippet: CD80 Antibody, anti-human, PE, REAfinityTM (Clone- REA661). Diluted 1:200 with dPBS by volume. , Miltenyi Biotec , Order Number: 130-123-253.

Techniques: Recombinant, Sterility, Modification, Staining, Saline, Multiplex Assay, Software

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