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: Chemical Science

Article Title: Bimodal accurate H 2 O 2 regulation to equalize tumor-associated macrophage repolarization and immunogenic tumor cell death elicitation †

doi: 10.1039/d4sc06305h

Figure Lengend Snippet: In vivo antitumor efficacy of ZnO 2 -ATM: inhibition of metastatic tumor growth and immune activation. (A) Primary tumor growth curves of various treatment groups. (B) Images of excised primary tumors after sacrificing mice at 14 days post-injection. (C) H&E-, TUNEL- and CRT-stained immunofluorescence of primary tumor sections at 1 day post-injection after ZnO 2 -ATM treatment. (D) Population changes of M1 (CD80 high CD206 low ) macrophages in primary tumors after various treatments at 3 days post-injection. (E) Population changes of maturing DCs (CD80 high CD86 high ) in spleens after various treatments at 3 days post-injection (gating on CD11c + ). (F) Distant tumor growth curves of various treatment groups. (G) Number of metastatic nodules in lungs after various treatments with the inset graph showing the corresponding lungs with metastatic nodules. (H) H&E-stained lung sections at 44 days post-injection. Red arrows indicate metastases.

Article Snippet: CRT antibodies, HMGB-1 antibodies, APC-conjugated CRT secondary antibodies and Alexa Fluor 594-conjugated HMGB secondary antibodies were purchased from Univ Biotech Co., Ltd. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Biofrox Co., Ltd. A TUNEL apoptosis detection kit (C1086) was purchased from Beyotime Biotechnology Co., Ltd. A TNF-α ELISA kit, an IL-6 ELISA kit, APC-conjugated anti-mouse CD80 (CD80-APC), FITC-conjugated anti-mouse CD11b (CD11b-FITC), FITC-conjugated anti-mouse CD11c (CD11c-FITC), PE-conjugated anti-mouse CD206 (CD206-PE), APC-conjugated anti-mouse CD3 (cat. 100236), PE-conjugated anti-mouse CD4 (CD4-PE), FITC-conjugated anti-mouse CD8a (CD8a-FITC), PE-conjugated anti-mouse CD86 (CD86-PE), and PE-conjugated anti-mouse CD44 (CD44-PE) were obtained from Multi-Science Co., Ltd. Annexin V-FITC/propidium iodide (PI) and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Yeasen Co., Ltd. Deionized water was generated using a Millipore Milli-Q system (Billerica, MA, USA).

Techniques: In Vivo, Inhibition, Activation Assay, Injection, TUNEL Assay, Staining, Immunofluorescence

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: Cell Reports Medicine

Article Title: Macrophages are activated toward phagocytic lymphoma cell clearance by pentose phosphate pathway inhibition

doi: 10.1016/j.xcrm.2024.101830

Figure Lengend Snippet:

Article Snippet: Rat monoclonal anti-CD86 , Miltenyi Biotec , Cat#130-123-724,;RRID: AB_2889634.

Techniques: Staining, Virus, Recombinant, Bicinchoninic Acid Protein Assay, Cell Viability Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Blocking Assay, Phospho-proteomics, Purification, Mass Spectrometry, Software, Modification