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Macrophages ingest β cell-derived dysfunctional mitochondria in the form of extracellular vesicles. (A) Generation of mtDsRed2-labeled MIN6 cells, cocultured with DIO-labeled BMDM for indicated time. (B) Representative confocal images of BMDM incubated with MIN6 cells (described in A) for 0.5, 12, and 24 h (n = 3). (C) Time-lapse confocal imaging revealing a mitochondria uptake event in macrophage. (D) qPCR analysis using specific primers for mtDNA from RAW264.7 <t>and</t> <t>β-TC6</t> (n = 3). (E) qPCR analysis of mtDNA from β-TC6 in RAW264.7 individual cultured (Mon-RAW264.7) or cocultured with β-TC6 (Co-RAW264.7) (n = 3). (F) Flow cytometry measured the transfer of mitochondria from Ins2p-mMito-DsRed2-labeled β cells to CD11b + F4/80 + macrophages isolated from mice islets and quantified the mean fluorescence intensity of mtDsRed2 (n = 6). (G) Representative confocal images of insulin immunofluorescence staining of mtDsRed2-labeled isolated islets (left). Flow cytometry measured mtDsRed2 in BMDM from the transwell system co-cultured with or without mtDsRed2-labeled isolated islets for 24 h (right) (n = 5). (H) The coculture and transwell culture systems determine the form of mitochondrial transfer. The mean fluorescence intensity of mtDsRed2 in RAW264.7 was analyzed by flow cytometry (n = 5). (I) Experimental schematic for collecting extracellular vesicles (EVs) from β cell-conditioned medium. (J) Proteomic analysis of mEVs. The six most representative cellular components are shown. Data are obtained from a pool of mEVs derived from β cells. (K) Representative confocal microscopy pictures of EVs from mtDsRed2-labeled β cells stained with DIO (n = 3). (L) Representative transmission electron microscopy image of EVs. The red arrow shows mitochondria in the EVs (n = 3). (M) Representative Western blot of Large EVs filtrated with 0.22 μm pore-size filter or not. CD63 and CD81 were used as markers of EVs, and TOM20 stands for mitochondria (n = 3). (N) Mitochondria membrane potential detection of β cells treated with or without PA and EVs from these β cells (n = 3). (O) Mitochondrial ROS content of EVs, from β cells treated with PA or not, was analyzed by MitoSOX staining (n = 6). Data are presented as the means ± SEMs. For D, E, F, G and O, statistical significance was calculated using Student's unpaired two-tailed t -test. For N, statistical significance was calculated using one-way ANOVA with Tukey's post hoc comparison. ∗∗ P < 0.01, ∗∗∗ P < 0.001.

β Tc6, supplied by Procell Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Islet regeneration protein Reg3g promotes macrophage clearance of β cell-derived dysfunctional mitochondria-rich vesicles to mitigate T2DM"

Article Title: Islet regeneration protein Reg3g promotes macrophage clearance of β cell-derived dysfunctional mitochondria-rich vesicles to mitigate T2DM

Journal: Redox Biology

doi: 10.1016/j.redox.2025.103996

Figure Legend Snippet: Macrophages ingest β cell-derived dysfunctional mitochondria in the form of extracellular vesicles. (A) Generation of mtDsRed2-labeled MIN6 cells, cocultured with DIO-labeled BMDM for indicated time. (B) Representative confocal images of BMDM incubated with MIN6 cells (described in A) for 0.5, 12, and 24 h (n = 3). (C) Time-lapse confocal imaging revealing a mitochondria uptake event in macrophage. (D) qPCR analysis using specific primers for mtDNA from RAW264.7 and β-TC6 (n = 3). (E) qPCR analysis of mtDNA from β-TC6 in RAW264.7 individual cultured (Mon-RAW264.7) or cocultured with β-TC6 (Co-RAW264.7) (n = 3). (F) Flow cytometry measured the transfer of mitochondria from Ins2p-mMito-DsRed2-labeled β cells to CD11b + F4/80 + macrophages isolated from mice islets and quantified the mean fluorescence intensity of mtDsRed2 (n = 6). (G) Representative confocal images of insulin immunofluorescence staining of mtDsRed2-labeled isolated islets (left). Flow cytometry measured mtDsRed2 in BMDM from the transwell system co-cultured with or without mtDsRed2-labeled isolated islets for 24 h (right) (n = 5). (H) The coculture and transwell culture systems determine the form of mitochondrial transfer. The mean fluorescence intensity of mtDsRed2 in RAW264.7 was analyzed by flow cytometry (n = 5). (I) Experimental schematic for collecting extracellular vesicles (EVs) from β cell-conditioned medium. (J) Proteomic analysis of mEVs. The six most representative cellular components are shown. Data are obtained from a pool of mEVs derived from β cells. (K) Representative confocal microscopy pictures of EVs from mtDsRed2-labeled β cells stained with DIO (n = 3). (L) Representative transmission electron microscopy image of EVs. The red arrow shows mitochondria in the EVs (n = 3). (M) Representative Western blot of Large EVs filtrated with 0.22 μm pore-size filter or not. CD63 and CD81 were used as markers of EVs, and TOM20 stands for mitochondria (n = 3). (N) Mitochondria membrane potential detection of β cells treated with or without PA and EVs from these β cells (n = 3). (O) Mitochondrial ROS content of EVs, from β cells treated with PA or not, was analyzed by MitoSOX staining (n = 6). Data are presented as the means ± SEMs. For D, E, F, G and O, statistical significance was calculated using Student's unpaired two-tailed t -test. For N, statistical significance was calculated using one-way ANOVA with Tukey's post hoc comparison. ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Techniques Used: Derivative Assay, Labeling, Incubation, Imaging, Cell Culture, Flow Cytometry, Isolation, Fluorescence, Immunofluorescence, Staining, Confocal Microscopy, Transmission Assay, Electron Microscopy, Western Blot, Pore Size, Membrane, Two Tailed Test, Comparison

Reg3g facilitates macrophage to clear β cell-derived mitochondria by mitophagy, lowering proinflammatory responses. (A) Contents of Heparan sulfate detection in RAW264.7 cells treated with Reg3g or not for 24 h in the presence of mEVs (n = 6). (B) Flow cytometry analysis of MIN6-derived mtDsRed2 in RAW264.7 treated with Reg3g or not in the presence of mEVs (n = 5). (C) qPCR analysis of mtDNA from β-TC6 in RAW264.7 treated with Reg3g under β-TC6-derived mEVs-stimulated condition (n = 3). (D) Flow cytometry analysis of β cell-derived mtDsRed2 in RAW264.7 after transfected with siExtl3 or mimic and treated with Reg3g or not in the presence of mEVs (n = 5). (E) qPCR analysis of mtDNA from β-TC6 in RAW264.7 after transfected with siExtl3 or mimic and treated with Reg3g under β-TC6-derived mEVs-stimulated condition (n = 3). (F–J) The RAW264.7 cells were treated with Reg3g or not in the presence of mEVs in these indicated experiments. (F) Immunofluorescent staining of LC3 + autophagosomes and co-localization with mtDsRed2 + in RAW264.7 (n = 3). (G) Co-localization of exogenous mtDsRed2 + mitochondria and Lyso-Tracker-labeled lysosome within RAW264.7 (n = 3). (H) Representative transmission electron microscopy image of RAW264.7. The red arrow shows mitophagy (n = 3). (I) Western blot analysis of mitophagy-related protein in RAW264.7 (n = 3). (J) RAW264.7 cells were transfected with AdPlus-mCherry-GFP-LC3B to detect the autophagosome formation (n = 3). (K–P) The BMDM cells were treated with Reg3g or not in the presence of mEVs in these indicated experiments. (K) Flow cytometry analysis of CD11c + and CD206 + cells ratios (n = 5). (L) mRNA levels of Il1b, Cd11c, Tnfα, iNos, Cd206, Ym1, Arg1 and Retnla were determined by RT-qPCR (n = 5). (M) Phagocytosis detection using FITC-Dextran (n = 5). (N) Contents of lactate detection (n = 5). (O) Extracellular acidification rate (ECAR) analysis in BMDMs (n = 6). (P) Oxygen consumption rate (OCR) analysis in BMDMs (n = 6). Data are presented as the means ± SEMs. For A, B, C, I, K, L, M, N, O and P, statistical significance was calculated using Student's unpaired two-tailed t -test. For D and E, statistical significance was calculated using ANOVA with Tukey's post hoc comparison. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Figure Legend Snippet: Reg3g facilitates macrophage to clear β cell-derived mitochondria by mitophagy, lowering proinflammatory responses. (A) Contents of Heparan sulfate detection in RAW264.7 cells treated with Reg3g or not for 24 h in the presence of mEVs (n = 6). (B) Flow cytometry analysis of MIN6-derived mtDsRed2 in RAW264.7 treated with Reg3g or not in the presence of mEVs (n = 5). (C) qPCR analysis of mtDNA from β-TC6 in RAW264.7 treated with Reg3g under β-TC6-derived mEVs-stimulated condition (n = 3). (D) Flow cytometry analysis of β cell-derived mtDsRed2 in RAW264.7 after transfected with siExtl3 or mimic and treated with Reg3g or not in the presence of mEVs (n = 5). (E) qPCR analysis of mtDNA from β-TC6 in RAW264.7 after transfected with siExtl3 or mimic and treated with Reg3g under β-TC6-derived mEVs-stimulated condition (n = 3). (F–J) The RAW264.7 cells were treated with Reg3g or not in the presence of mEVs in these indicated experiments. (F) Immunofluorescent staining of LC3 + autophagosomes and co-localization with mtDsRed2 + in RAW264.7 (n = 3). (G) Co-localization of exogenous mtDsRed2 + mitochondria and Lyso-Tracker-labeled lysosome within RAW264.7 (n = 3). (H) Representative transmission electron microscopy image of RAW264.7. The red arrow shows mitophagy (n = 3). (I) Western blot analysis of mitophagy-related protein in RAW264.7 (n = 3). (J) RAW264.7 cells were transfected with AdPlus-mCherry-GFP-LC3B to detect the autophagosome formation (n = 3). (K–P) The BMDM cells were treated with Reg3g or not in the presence of mEVs in these indicated experiments. (K) Flow cytometry analysis of CD11c + and CD206 + cells ratios (n = 5). (L) mRNA levels of Il1b, Cd11c, Tnfα, iNos, Cd206, Ym1, Arg1 and Retnla were determined by RT-qPCR (n = 5). (M) Phagocytosis detection using FITC-Dextran (n = 5). (N) Contents of lactate detection (n = 5). (O) Extracellular acidification rate (ECAR) analysis in BMDMs (n = 6). (P) Oxygen consumption rate (OCR) analysis in BMDMs (n = 6). Data are presented as the means ± SEMs. For A, B, C, I, K, L, M, N, O and P, statistical significance was calculated using Student's unpaired two-tailed t -test. For D and E, statistical significance was calculated using ANOVA with Tukey's post hoc comparison. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Techniques Used: Derivative Assay, Flow Cytometry, Transfection, Staining, Labeling, Transmission Assay, Electron Microscopy, Western Blot, Quantitative RT-PCR, Two Tailed Test, Comparison

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Journal: Redox Biology

Article Title: Islet regeneration protein Reg3g promotes macrophage clearance of β cell-derived dysfunctional mitochondria-rich vesicles to mitigate T2DM

doi: 10.1016/j.redox.2025.103996

Figure Lengend Snippet: Macrophages ingest β cell-derived dysfunctional mitochondria in the form of extracellular vesicles. (A) Generation of mtDsRed2-labeled MIN6 cells, cocultured with DIO-labeled BMDM for indicated time. (B) Representative confocal images of BMDM incubated with MIN6 cells (described in A) for 0.5, 12, and 24 h (n = 3). (C) Time-lapse confocal imaging revealing a mitochondria uptake event in macrophage. (D) qPCR analysis using specific primers for mtDNA from RAW264.7 and β-TC6 (n = 3). (E) qPCR analysis of mtDNA from β-TC6 in RAW264.7 individual cultured (Mon-RAW264.7) or cocultured with β-TC6 (Co-RAW264.7) (n = 3). (F) Flow cytometry measured the transfer of mitochondria from Ins2p-mMito-DsRed2-labeled β cells to CD11b + F4/80 + macrophages isolated from mice islets and quantified the mean fluorescence intensity of mtDsRed2 (n = 6). (G) Representative confocal images of insulin immunofluorescence staining of mtDsRed2-labeled isolated islets (left). Flow cytometry measured mtDsRed2 in BMDM from the transwell system co-cultured with or without mtDsRed2-labeled isolated islets for 24 h (right) (n = 5). (H) The coculture and transwell culture systems determine the form of mitochondrial transfer. The mean fluorescence intensity of mtDsRed2 in RAW264.7 was analyzed by flow cytometry (n = 5). (I) Experimental schematic for collecting extracellular vesicles (EVs) from β cell-conditioned medium. (J) Proteomic analysis of mEVs. The six most representative cellular components are shown. Data are obtained from a pool of mEVs derived from β cells. (K) Representative confocal microscopy pictures of EVs from mtDsRed2-labeled β cells stained with DIO (n = 3). (L) Representative transmission electron microscopy image of EVs. The red arrow shows mitochondria in the EVs (n = 3). (M) Representative Western blot of Large EVs filtrated with 0.22 μm pore-size filter or not. CD63 and CD81 were used as markers of EVs, and TOM20 stands for mitochondria (n = 3). (N) Mitochondria membrane potential detection of β cells treated with or without PA and EVs from these β cells (n = 3). (O) Mitochondrial ROS content of EVs, from β cells treated with PA or not, was analyzed by MitoSOX staining (n = 6). Data are presented as the means ± SEMs. For D, E, F, G and O, statistical significance was calculated using Student's unpaired two-tailed t -test. For N, statistical significance was calculated using one-way ANOVA with Tukey's post hoc comparison. ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The mouse pancreatic β cell lines MIN6 (RRID:CVCL_0431) and β-TC6 (RRID:CVCL_0605) were purchased from Procell Life Science & Technology Co. Ltd. (Wuhan, China) and maintained in DMEM containing 10 % (for MIN6)/15 % (for β-TC6) FBS, and 1 % penicillin-streptomycin.

Techniques: Derivative Assay, Labeling, Incubation, Imaging, Cell Culture, Flow Cytometry, Isolation, Fluorescence, Immunofluorescence, Staining, Confocal Microscopy, Transmission Assay, Electron Microscopy, Western Blot, Pore Size, Membrane, Two Tailed Test, Comparison

Journal: Redox Biology

Article Title: Islet regeneration protein Reg3g promotes macrophage clearance of β cell-derived dysfunctional mitochondria-rich vesicles to mitigate T2DM

doi: 10.1016/j.redox.2025.103996

Figure Lengend Snippet: Reg3g facilitates macrophage to clear β cell-derived mitochondria by mitophagy, lowering proinflammatory responses. (A) Contents of Heparan sulfate detection in RAW264.7 cells treated with Reg3g or not for 24 h in the presence of mEVs (n = 6). (B) Flow cytometry analysis of MIN6-derived mtDsRed2 in RAW264.7 treated with Reg3g or not in the presence of mEVs (n = 5). (C) qPCR analysis of mtDNA from β-TC6 in RAW264.7 treated with Reg3g under β-TC6-derived mEVs-stimulated condition (n = 3). (D) Flow cytometry analysis of β cell-derived mtDsRed2 in RAW264.7 after transfected with siExtl3 or mimic and treated with Reg3g or not in the presence of mEVs (n = 5). (E) qPCR analysis of mtDNA from β-TC6 in RAW264.7 after transfected with siExtl3 or mimic and treated with Reg3g under β-TC6-derived mEVs-stimulated condition (n = 3). (F–J) The RAW264.7 cells were treated with Reg3g or not in the presence of mEVs in these indicated experiments. (F) Immunofluorescent staining of LC3 + autophagosomes and co-localization with mtDsRed2 + in RAW264.7 (n = 3). (G) Co-localization of exogenous mtDsRed2 + mitochondria and Lyso-Tracker-labeled lysosome within RAW264.7 (n = 3). (H) Representative transmission electron microscopy image of RAW264.7. The red arrow shows mitophagy (n = 3). (I) Western blot analysis of mitophagy-related protein in RAW264.7 (n = 3). (J) RAW264.7 cells were transfected with AdPlus-mCherry-GFP-LC3B to detect the autophagosome formation (n = 3). (K–P) The BMDM cells were treated with Reg3g or not in the presence of mEVs in these indicated experiments. (K) Flow cytometry analysis of CD11c + and CD206 + cells ratios (n = 5). (L) mRNA levels of Il1b, Cd11c, Tnfα, iNos, Cd206, Ym1, Arg1 and Retnla were determined by RT-qPCR (n = 5). (M) Phagocytosis detection using FITC-Dextran (n = 5). (N) Contents of lactate detection (n = 5). (O) Extracellular acidification rate (ECAR) analysis in BMDMs (n = 6). (P) Oxygen consumption rate (OCR) analysis in BMDMs (n = 6). Data are presented as the means ± SEMs. For A, B, C, I, K, L, M, N, O and P, statistical significance was calculated using Student's unpaired two-tailed t -test. For D and E, statistical significance was calculated using ANOVA with Tukey's post hoc comparison. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Techniques: Derivative Assay, Flow Cytometry, Transfection, Staining, Labeling, Transmission Assay, Electron Microscopy, Western Blot, Quantitative RT-PCR, Two Tailed Test, Comparison

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