Journal: Cell metabolism

Article Title: Ejection of damaged mitochondria and their removal by macrophages ensure efficient thermogenesis in brown adipose tissue

doi: 10.1016/j.cmet.2022.02.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD86 Antibody, anti-mouse, FITC, Miltenyi Biotec , Miltenyi Biotec , Cat# 130–123-672; RRID:AB_2889633.

Techniques: Immunofluorescence, Recombinant, Cytometry, Purification, shRNA, Cell Culture, Lysis, XF Assay, Staining, Isolation, Transfection, Live Cell Imaging, Mouse Assay, Software, Expressing

Journal: Journal of leukocyte biology

Article Title: Adipose tissue of human omentum is a major source of dendritic cells, which lose MHC Class II and stimulatory function in Crohn's disease.

doi: 10.1189/jlb.0905501

Figure Lengend Snippet: Fig. 5. Morphology of omental DC. (a) A typical DC from a control omentum is shown. (b) An example of immunogold staining of the veils of a DC. The original bars indicate 1 m. In controls, gold labeling was always 5 gold grains per cell; 10 grains was considered positive. (c) The types of myeloid cells identified in the omental cells from two control patients; the proportion positive for HLA-DR staining is shown in the solid portion of the histograms, and those negative are hatched. M , Macrophage. (d) The gold grains per cell for the surface DR-labeling in the two experiments performed are shown. No evidence of DR-labeling was seen in either of the two Crohn’s patients studied. (e) A light microscopy picture of a cluster of putative DC immunostained with CD11c on a frozen section of omentum from a Crohn’s patient showing the dark, positive-peroxidase staining, which was absent from control slides. Cell nuclei, which were stained with hematoxylin, were distinguishable, showing that the veils were associated with mononuclear cells in the section. (f) Control for e using serum instead of specific antibody and with only hematoxylin staining visible. (g) Frozen section of control omentum, showing two cells specifically labeled for CD83 (TRITC-labeled). (h) The same section as g with label specific for CD86 (FITC-labeled).

Article Snippet: To block nonspecific binding sites, sections were incubated with normal mouse serum for 1 h and then incubated with FITC-conjugated primary antibody to CD86, CD80, or CD40 (Serotec) for 4 h, washed in PBS, and mounted under cover-slips using 4 ,6-diamidino-2-phenylindole-conjugated mounting medium (Vector Laboratories).

Techniques: Control, Staining, Labeling, Light Microscopy

Journal: Pharmaceutics

Article Title: Development and Evaluation of a Novel Diammonium Glycyrrhizinate Phytosome for Nasal Vaccination

doi: 10.3390/pharmaceutics14102000

Figure Lengend Snippet: Flow cytometric analysis of CD11c, CD80, and CD86 expression in BMDCs after treatment of blank medium, free DG, and DG-P for 24 h ( n = 4).

Article Snippet: Fluorochrome-labelled anti-mouse monoclonal antibodies including APC-CD11c, FITC-CD80, and PE-CD86, were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Expressing

Journal: Pharmaceutics

Article Title: Development and Evaluation of a Novel Diammonium Glycyrrhizinate Phytosome for Nasal Vaccination

doi: 10.3390/pharmaceutics14102000

Figure Lengend Snippet: Quantification of CD80 and CD86 expression on BMDCs after activation ( n = 4). Note: *, ** and *** represent the differences at p < 0.05, p < 0.01, and p < 0.001, respectively.

Article Snippet: Fluorochrome-labelled anti-mouse monoclonal antibodies including APC-CD11c, FITC-CD80, and PE-CD86, were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Expressing, Activation Assay

Journal: Science Advances

Article Title: Biofunctional lipid nanoparticles for precision treatment and prophylaxis of bacterial infections

doi: 10.1126/sciadv.adk9754

Figure Lengend Snippet: ( A ) Expression levels of CD80 + and CD86 + in BMDCs (CD11c + ) and ( B ) production of IL-6 and TNF-α of the cell supernatant measured by ELISA after different treatments (concentration, 2 μg ml −1 NPs) for 24 hours ( n = 3). ( C ) Schematics of experimental design to evaluate the in vivo short-term immune responses activated by a single dose of various LNPs (20 μg/20 g) via subcutaneous injection. ( D ) Percentage of CD80 + and ( E ) CD86 + cells (gated on CD11c + cells) in the LNs at day 3 after immunization ( n = 6). ( F ) Percentage of CD3 + T cells, ( G ) CD8 + T cells (gated on CD3 + T cells), and ( H ) CD4 + T cells (gated on CD3 + T cells) in the spleen at day 3 after immunization ( n = 6). Naive mice without immunization were used as control. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Fluorescein isothiocyanate (FITC)–conjugated anti-mouse CD11c, phycoerythrin (PE)–conjugated anti-mouse CD80, PE-conjugated anti-mouse CD86, allophycocyanin (APC)–conjugated anti-mouse CD3, PE-conjugated anti-mouse CD8a, and FITC-conjugated anti-mouse CD4 were purchased from Tonbo Biosciences, USA.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, In Vivo, Injection, Control

Journal: International Journal of Molecular Sciences

Article Title: TNFAIP3 Deficiency Affects Monocytes, Monocytes-Derived Cells and Microglia in Mice

doi: 10.3390/ijms21082830

Figure Lengend Snippet: Flow-cytometry gating strategy used for spleen and lymph nodes. ( A ) Gating strategy for lymphocytes, NK and B cells; (1) FS versus SS gating was used to discriminate cells based on their size; (2) living cells were then identified by propidium iodide negativity; (3) CD3 was used as a T-cell marker; (4) CD3+ cells were distinguished based on the presence of CD4 and CD8; (5) NK and NK T were distinguished based on the presence of CD49b; (6) CD45R was used as a B-cell marker. ( B ) Gating strategy for macrophages, monocytes and DCs; (1) FS versus SS gating was used to discriminate cells based on their size; (2) living cells were then identified by propidium iodide negativity; (3–4) F4/80 gating on CD11b+ cells was used to select macrophages; (5) Ly6-C and Ly6-G gating on CD11b+ cells was used to select monocytes; (6) DCs were distinguished based on the presence of CD11c and CD86. (NK, natural killer; FS, forward scatter; SS side scatter; DCs, dendritic cells).

Article Snippet: The following anti-mouse mAbs were used: CD3-allophycocyanin (APC, 130-109-838), CD45R-B220-phycoerythrin (PE, 130-102-292), CD49b-fluorescein isothiocyanate (FITC, 130-102-258), CD11c-APC (130-102-493), F4/80-APC (130-102-379), CD86-PE (130-102-604), c-kit-(CD117)-PE (130-111-693), CD16/CD32-PE (130-102-429) (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD4-phycoerythrin-cy7 (PECy7, BMS25-0041-82), CD8-fluorescein isothiocyanate (FITC, BMS11-0081-82), CD11b-PE (BMS12-0112-82), Ly6-C-AlexaFluo488 (BMS53-5932-82), Ly6-G-APC (BMS17-5931-82), Lineage cocktail (Lin)-PerCP-Cy5.5 (BD Pharmingen, 51-9006964, San Jose, CA, US), Sca1-APC (130-106-425), CD34-FITC (130-105-831) (eBioscience, ThermoFisher Scientific, San Diego, CA, US).

Techniques: Flow Cytometry, Marker

Journal: International Journal of Molecular Sciences

Article Title: TNFAIP3 Deficiency Affects Monocytes, Monocytes-Derived Cells and Microglia in Mice

doi: 10.3390/ijms21082830

Figure Lengend Snippet: Flow-cytometry analysis performed on spleen obtained from 3 month-old WT and TNFAIP3 cx3cr1-KO mice. The analysis reveals that the percentage number of CD11b + F4/80 + macrophages ( A , p = 0.004), CD11b + Ly-6C + Ly-6G + monocytes ( B , p = 0.016), CD11c + CD86 + DCs ( C , p = 0.048) and B200 + B ( F , p = 0.048) cells is reduced in TNFAIP3 cx3cr1-KO mice compared to their WT littermates. No differences are reported in CD49 + NK ( D , p = 0.214) and CD3 + CD49 + NK T cells ( E , p = 0.153) CD3 + T ( G , p = 0.367), CD3 + CD4 + T helper ( H , p = 0.683) and CD3 + CD8 + cytotoxic T cells ( I , p = 0.109) (WT n = 8; TNFAIP3 cx3cr1-KO n = 4) (Mann–Whitney test, ** p < 0.01; * p < 0.05). (DCs, dendritic cells; NK, natural killer).

Article Snippet: The following anti-mouse mAbs were used: CD3-allophycocyanin (APC, 130-109-838), CD45R-B220-phycoerythrin (PE, 130-102-292), CD49b-fluorescein isothiocyanate (FITC, 130-102-258), CD11c-APC (130-102-493), F4/80-APC (130-102-379), CD86-PE (130-102-604), c-kit-(CD117)-PE (130-111-693), CD16/CD32-PE (130-102-429) (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD4-phycoerythrin-cy7 (PECy7, BMS25-0041-82), CD8-fluorescein isothiocyanate (FITC, BMS11-0081-82), CD11b-PE (BMS12-0112-82), Ly6-C-AlexaFluo488 (BMS53-5932-82), Ly6-G-APC (BMS17-5931-82), Lineage cocktail (Lin)-PerCP-Cy5.5 (BD Pharmingen, 51-9006964, San Jose, CA, US), Sca1-APC (130-106-425), CD34-FITC (130-105-831) (eBioscience, ThermoFisher Scientific, San Diego, CA, US).

Techniques: Flow Cytometry, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: TNFAIP3 Deficiency Affects Monocytes, Monocytes-Derived Cells and Microglia in Mice

doi: 10.3390/ijms21082830

Figure Lengend Snippet: Flow-cytometry analysis performed on lymph nodes obtained from 3 month-old WT and TNFAIP3 cx3cr1-KO mice. The analysis reveals that the percentage number of CD11b + F4/80 + macrophages ( A , p = 0.006), CD49 + NK ( D , p = 0.009) and CD3 + CD49 + NK T ( E , p = 0.009) cells is reduced in TNFAIP3 cx3cr1-KO mice compared to their WT littermates. No differences are reported in CD11b + Ly-6C + Ly-6G + monocytes ( B , p = 0.914), CD3 + T ( G , p = 0.114) and CD3 + CD4 + T helper cells ( H , p = 0.066). The percentage number of CD11c + CD86 + DCs ( C , p = 0.019), B200 + B ( F , p = 0.019) and CD3 + CD8 + cytotoxic T ( I , p = 0.009) cells is increased in TNFAIP3 cx3cr1-KO mice compared to their WT littermates. (WT n = 8; TNFAIP3 cx3cr1-KO n = 4) (Mann–Whitney test, ** p < 0.01; * p < 0.05). (DCs, dendritic cells; NK, natural killer).

Article Snippet: The following anti-mouse mAbs were used: CD3-allophycocyanin (APC, 130-109-838), CD45R-B220-phycoerythrin (PE, 130-102-292), CD49b-fluorescein isothiocyanate (FITC, 130-102-258), CD11c-APC (130-102-493), F4/80-APC (130-102-379), CD86-PE (130-102-604), c-kit-(CD117)-PE (130-111-693), CD16/CD32-PE (130-102-429) (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD4-phycoerythrin-cy7 (PECy7, BMS25-0041-82), CD8-fluorescein isothiocyanate (FITC, BMS11-0081-82), CD11b-PE (BMS12-0112-82), Ly6-C-AlexaFluo488 (BMS53-5932-82), Ly6-G-APC (BMS17-5931-82), Lineage cocktail (Lin)-PerCP-Cy5.5 (BD Pharmingen, 51-9006964, San Jose, CA, US), Sca1-APC (130-106-425), CD34-FITC (130-105-831) (eBioscience, ThermoFisher Scientific, San Diego, CA, US).

Techniques: Flow Cytometry, MANN-WHITNEY

Journal: bioRxiv

Article Title: Therapeutic poxviruses induce the secretion of immunostimulating and anti-tumoral extracellular vesicles

doi: 10.1101/2025.09.19.677320

Figure Lengend Snippet: A) Workflow for assessing the presence of cellular proteins (CD63, ICAM1 and CD86) and virus-encoded therapeutic payloads (IL-12, CD40L and OVA peptide SIINFEKL presented on MHCI) on EVs secreted by mouse dendritic cells (DC2.4) and isolated by ultracentrifugation (UC) or size exclusion chromatography (SEC; EVs-rich fractions (EVs SEC, fractions 1-4) and soluble proteins-rich fractions (Prots SEC, fractions 5-10)) following 100nm filtration. Payloads were quantified by electrochemiluminescence immunoassay (B-G) on EVs isolated from non-infected cells (mock) or from cells infected with empty or armed MVA virus (encoding IL12, CD40L and OVA peptide SIINFEKL payloads). B) Quantification of CD63 on EVs. EVs mock Vs EVs empty virus p=0,0007; EVs mock Vs EVs armed virus UC p= 0,0001; EVs mock Vs EVs armed virus SEC p< 0,0001; EVs mock Vs Soluble proteins armed virus SEC p< 0,0001; One-way Anova; N=5 independent experiments. C) Quantification of ICAM1 on EVs. EVs mock Vs EVs empty virus: p=0,0276; EVs mock Vs EVs armed virus UC p= 0,0012; EVs mock Vs EVs armed virus SEC p= 0,0425; EVs mock Vs Soluble proteins armed virus SEC p= 0,83; One-way Anova; N=4 independent experiments. D) Quantification of CD86 on EVs. EVs mock Vs EVs empty virus: p=0,19; EVs mock Vs EVs armed virus UC p>0,99; EVs mock Vs EVs armed virus SEC p= 0,73; EVs mock Vs Soluble proteins armed virus SEC p= 0,99; Kruskal-Wallis; N=4 independent experiments. E) Quantification of CD40L on EVs. EVs mock Vs EVs empty virus: p>0,99; EVs mock Vs EVs armed virus UC p<0,0001; EVs mock Vs EVs armed virus SEC p= 0,011; EVs mock Vs Soluble proteins armed virus SEC p>0,99; EVs armed virus SEC Vs Soluble proteins armed virus SEC p=0,0125; One-way Anova; N=4 independent experiments. F) Quantification of MHCI bound OVA peptide (SIINFEKL). EVs mock Vs EVs empty virus: p>0,99; EVs mock Vs EVs armed virus UC p=0,023; EVs mock Vs EVs armed virus SEC p= 0,0341; EVs mock Vs Soluble proteins armed virus SEC p>0,99; Kruskal-Wallis; N=4 independent experiments. E) Quantification of IL-12. EVs mock Vs EVs empty virus p=0,045; EVs mock Vs EVs armed virus UC p>0,99; EVs mock Vs Soluble proteins armed virus SEC p=0,0004; EVs armed virus SEC Vs Soluble proteins armed virus SEC p=0,045; Kruskal-Wallis test. N=5 independent experiments. * p<0,05; ** p<0,01; *** p<0,005; **** p<0,001; ***** p<0,0001; ns: non-significant.

Article Snippet: The following detection antibodies were used: CD63 Antibody, anti-mouse Biotin (clone REA563, Miltenyi Biotec); CD54 (ICAM-1) Antibody, anti-mouse, Biotin (clone YN1/1.7.4, #130-104-213, Miltenyi Biotec); CD86 Antibody, anti-mouse, Biotin (clone PO3.3, #130-101-944, Miltenyi Biotec); CD154 (CD40L) Antibody, anti-mouse, Biotin (clone MR1, #130-101-900, Miltenyi Biotec); H-2Kb/SIINFEKL Antibody, anti-mouse Biotin (clone 25-D1.16, Miltenyi Biotec).

Techniques: Virus, Isolation, Size-exclusion Chromatography, Filtration, Electrochemiluminescence, Infection

Journal: Acta Pharmaceutica Sinica. B

Article Title: Novel Pt(IV) complex OAP2 induces STING activation and pyroptosis via mitochondrial membrane remodeling for synergistic chemo-immunotherapy

doi: 10.1016/j.apsb.2023.11.032

Figure Lengend Snippet: The immune activation effect of OAP2 in vivo . (A–B) Analysis of mature DC cells in mouse spleen. Expression of CD80 and CD86 quantitatively detected by flow cytometry (A), and statistical analysis (B). (C–D) Analysis of mature DC cells in mouse tumor site. Expression of CD80 and CD86 quantitatively detected by flow cytometry (C), and statistical analysis (D). (E–F) Analysis of cytotoxic T cells in mouse tumor. Expression of CD4 and CD8 quantitatively detected by flow cytometry (E), and statistical analysis (F). (G–N) Tumor tissue cytokine expression content measured by ELISA ( n = 3).

Article Snippet: The following antibodies and kits were used in the flow cytometry and ELISA analyses: CoraLite® Plus 488 Anti-Mouse CD4, CoraLite®568 Anti-Mouse CD3, APC Anti-Mouse CD86, FITC Plus Anti-Mouse CD11c, and PE Anti-Mouse CD80 (B7-1) were purchased from Proteintech.

Techniques: Activation Assay, In Vivo, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay