cd63  (Developmental Studies Hybridoma Bank)

Journal: The Journal of Cell Biology

Article Title: Rsp5/NEDD4 and ESCRT regulate TDP-43 toxicity and turnover via an endolysosomal clearance mechanism

doi: 10.1083/jcb.202212064

Figure Lengend Snippet: Giant Rab7 vesicular structures exhibit CD63 colocalization and CHQ-sensitive intraluminal CD63 bodies, suggesting an endolysosomal origin. (A) HEK293A cells expressing endogenous TDP-43 (WT) or TDP-43-GFP (pseudocolored white in merge) were immunostained for Rab7 (pseudocolored red) and CD63 (pseudocolored green). Percentages in zoom panels indicate Rab7 foci that overlap with CD63 foci. (B) TDP-43-GFP (pseudocolored white) expressing cells were immunostained for Rab7 (pseudocolored red in merge) and CD63 (pseudocolored green in merge) in the presence or absence of CHQ. Inclusion of multiple rows reflects diverse phenotypes observed. Green arrowheads indicate CD63 foci on the intraluminal Rab7 vesicular membrane (top row) or fully within Rab7 vesicles (second and fourth row). Red arrowheads indicate Rab7 puncta fully within Rab 7 vesicles (second row). White arrowheads indicate TDP-43 puncta within Rab7 vesicles (second and fourth row; bottom right in latter case), on the external Rab7 vesicular membrane (fifth row), or in distinct cytoplasmic puncta (fourth and sixth row). Scale bar, 10 µm. (C) Quantification of CD63 signal (average and by bins) within the lumen of Rab7 vesicles. *, P < 0.05 by Mann–Whitney U test. (D) Average Rab7 vesicle lumen area ± CHQ. ***, P < 0.001 by Mann–Whitney U test. (E) Percentage of TDP-43 localization phenotypes relative to Rab7 and CD63, binned by category. Error bars in all graphical panels represent standard error of the mean.

Article Snippet: Primary antibodies used for the immunofluorescence were Rab7 (ab137029; Abcam, dilution: 1:200), CD63 (H5C6; Developmental Studies Hybridoma Bank, dilution 1:100), Rab5 (ab109534; Abcam, dilution: 1:200), LC3B (3868S; Cell Signaling, dilution 1:500), LAMP1 (21997-1-AP; Proteintech, dilution 1:200), and TDP-43 (10782-2-AP; Proteintech; dilution 1:200).

Techniques: Expressing, Membrane, MANN-WHITNEY

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: Antibodies used to determine protein expression list as follows: CD63 (Sc-5275, Santa), CD81 (HY– P80608 , MCE), LC3A/B (#12741, CST; #AF5402, Affinity Biosciences), TOM20 (66777-1-Ig, Proteintech), P2RX7 (28207-1-AP, Proteintech), NF-κB (#8242, CST), p–NF–κB (#3033, CST).

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: 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: Antibodies used to determine protein expression list as follows: CD63 (Sc-5275, Santa), CD81 (HY– P80608 , MCE), LC3A/B (#12741, CST; #AF5402, Affinity Biosciences), TOM20 (66777-1-Ig, Proteintech), P2RX7 (28207-1-AP, Proteintech), NF-κB (#8242, CST), p–NF–κB (#3033, CST).

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: Cells

Article Title: Comparative Profiling of Mouse and Human Microglial Small Extracellular Vesicles Reveals Conserved Core Functions with Distinct miRNA Signatures

doi: 10.3390/cells15020184

Figure Lengend Snippet: Robust expression exhibiting a comparative CD63-tdTomato-labeled vesicle load in resting microglia across species. ( A – C ) Confocal microscopy images of immortalized human microglia in resting state showing the presence of CD63-tdTomato-labeled vesicles (red). Cells were counterstained with Alexa Fluor™ 488 Phalloidin (green) to visualize microglial cell bodies and Hoechst (blue) to label nuclei. ( A , B ) Low-magnification view showing widespread intracellular distribution of CD63-tdTomato-labeled with majority exhibiting a perinuclear accumulation. Higher-magnification (40×) image of HuMG parent cells ( C ) demonstrating punctate CD63-positive vesicles localized along actin filaments and concentrated in the perinuclear region. ( D , E ) Resting mouse microglia exhibiting robust expression of CD63-tdTomato-labeled intracellular vesicles. ( D ) Phalloidin-488 staining defines cell boundaries and morphology, revealing widespread CD63-positive puncta throughout the cytoplasm. ( E ) Hoechst counterstaining confirms intracellular vesicle localization surrounding the nucleus. ( F ) Quantification of intracellular vesicle load in resting microglia of both mouse and human origin displayed significantly higher cellular load of CD63-expressing intracellular vesicles in microglia of mouse origin compared to that of human origin (** p < 0.01). Bars represent mean ± SEM; acquired from images obtained from independent biological replicates from each species. Scale bar: 15 µm ( A , B , D , E ) and 5 µm ( C ).

Article Snippet: Parent human (HMC3) and mouse (BV2) microglial cells stably expressing an sEV reporter were generated by transduction with a lentiviral vector encoding a CD63-tdTomato fusion (LentifectTM Purified Lentiviral Particles, Cat. # LP772-025, Genecopoeia, Rockville, MD, USA) and cultured to ~90% confluence.

Techniques: Expressing, Labeling, Confocal Microscopy, Staining

Journal: Cells

Article Title: Comparative Profiling of Mouse and Human Microglial Small Extracellular Vesicles Reveals Conserved Core Functions with Distinct miRNA Signatures

doi: 10.3390/cells15020184

Figure Lengend Snippet: Time-dependent uptake of CD63-tdTomato-labeled MGEVs by HuSCs. ( A – F ) Confocal images of HuSC exposed to purified CD63-tdTomato-labeled MsMGEVs (red) for 24 h ( A , B ), 48 h ( C , D ), or 72 h ( E , F ). Cells were stained with Alexa Fluor™ 488 Phalloidin (green) to mark the cell body and Hoechst to label nuclei (blue). Internalized CD63-tdTomato-positive sEVs appear as red puncta distributed throughout the cytoplasm and concentrated in perinuclear regions. The size of fluorescent puncta reflects intracellular endosomal accumulation of multiple internalized sEVs rather than aggregation of individual vesicles. Uptake of MsMGEVs showed a trend in an increase in sEVs overtime. Confocal images of HuSC treated with purified CD63-tdTomato-labeled HuMGEVs, red) for 24 h ( G , H ), 48 h ( I , J ), or 72 h ( K , L ). Like MsMGEVs, HuMGEV uptake was evident as intracellular red puncta; however, the overall accumulation pattern remained relatively stable across time points. ( M , N ) shows untreated HuSC microglia (control with no MGEV exposure) showing phalloidin-488 and Hoechst staining but no detectable CD63-tdTomato signal, confirming that red puncta in treated groups represent internalized CD63-tdTomato-labeled MGEVs. ( O ) Quantification of CD63-tdTomato fluorescence intensity in HuSC cells following 24 h, 48 h, or 72 h exposure to MsMGEVs or HuMGEVs. Both vesicle types were taken up by HuSC microglia, with MsMGEVs showing a modest trend toward increased accumulation over time. Bars represent mean ± SEM; individual points represent biological replicates. Scale Bar: 15 µm.

Article Snippet: Parent human (HMC3) and mouse (BV2) microglial cells stably expressing an sEV reporter were generated by transduction with a lentiviral vector encoding a CD63-tdTomato fusion (LentifectTM Purified Lentiviral Particles, Cat. # LP772-025, Genecopoeia, Rockville, MD, USA) and cultured to ~90% confluence.

Techniques: Labeling, Purification, Staining, Control, Fluorescence

Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Lentiviral transduction to generate WT-, PanKO-, CD9KO-, CD63KO-, and CD81KO-cells expressing TlucCD9-Cerulean. A. Schematic workflow of generating engineered Tluc-EVs by introducing TlucCD9-Cerulean lentiviruses (created with BioRender.com ). B. The percentage of Cerulean positive cells. C. The mean fluorescence intensity (MFI) of Cerulean positive cells. D. Flow cytometry plot for the cells after staining with APC-conjugated CD9/CD63/CD81 tetraspanin antibodies. E. Fold increase in engineered Tluc-CD9 EVs in PanKO-, CD9KO, CD63KO-, and CD81KO-cells over WT cells (Data are normalized to the RLU of EVs from WT cells). F. Fold increase of engineered Tluc-CD9EVs over PanKO-, CD9KO, CD63KO-, and CD81KO-cells (Data are normalized to the RLU of EVs from WT cells and compared across KO groups). G. Heatmap of tetraspanins in EVs. H. Interaction network of CD9 with the tetraspanins retrieved from STRING. The data are presented as means (±SD, n = 3-5). One-way ANOVA was used to show significance and was illustrated as follows: **** p < 0.0001.

Article Snippet: 50 μl of purified EVs at a concentration of 1 × 10 10 /ml were stained with either anti-human CD9 (Miltenyi Biotech, clone SN4), anti-human CD63 (Miltenyi Biotec, clone H5C6) and anti-human CD81 antibodies (Beckman Coulter, clone JS64) or REA control APC conjugated antibodies at a concentration of 8 nM overnight at room temperature in the dark.

Techniques: Transduction, Expressing, Fluorescence, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Lentiviral transduction to generate WT, PanKO, CD9KO, CD63KO, and CD81KO cells expressing TlucCD63-Cerulean. A. Schematic workflow of engineered Tluc-EVs by introducing TlucCD63-Cerulean lentiviruses (created with BioRender.com ). B. The percentage of Cerulean positive cells. C. The mean fluorescent intensity (MFI) of Cerulean positive cells. D. Flow cytometry plot for the cells after staining with APC-conjugated CD9/CD63/CD81 tetraspanin antibodies. E. Fold increase of engineered Tluc-CD63 EVs in PanKO-, CD9KO, CD63KO-, and CD81KO-cells over WT cells (Data are normalized to the RLU of EVs from WT cells). F. Fold increase of engineered Tluc-CD63EVs in between PanKO-, CD9KO, CD63KO-, and CD81KO-cells (Data are normalized to the RLU of EVs from WT cells and compared across KO groups). G. Heatmap of expressing tetraspanins in EVs. H. Interaction network of CD63 with the tetraspanins retrieved from STRING. The data are presented as means (±SD, n = 3-5). One-way ANOVA was used to show significance and was illustrated as follows: * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: 50 μl of purified EVs at a concentration of 1 × 10 10 /ml were stained with either anti-human CD9 (Miltenyi Biotech, clone SN4), anti-human CD63 (Miltenyi Biotec, clone H5C6) and anti-human CD81 antibodies (Beckman Coulter, clone JS64) or REA control APC conjugated antibodies at a concentration of 8 nM overnight at room temperature in the dark.

Techniques: Transduction, Expressing, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Lentiviral transduction to generate WT-, PanKO-, CD9KO-, CD63KO-, and CD81KO-cells expressing TlucCD9-Cerulean. A. Schematic workflow of engineered Tluc-EVs by introducing TlucCD81-Cerulean lentiviruses (created with BioRender.com ). B. The percentage of Cerulean positive cells. C. The MFI of Cerulean positive cells. D. Flow cytometry plot for the cells after staining with APC-conjugated CD9/CD63/CD81 tetraspanin antibodies. E. Fold increase of engineered Tluc-CD81 EVs in PanKO-, CD9KO, CD63KO-, and CD81KO-cells over WT cells (Data are normalized to the RLU of EVs from WT cells). F. Fold increase of engineered Tluc-CD81EVs in between PanKO-, CD9KO, CD63KO-, and CD81KO-cells (Data are normalized to the RLU of EVs from WT cells and compared across KO groups). G. Heatmap of expressing tetraspanins in EVs. H. Interaction network of CD81 with the tetraspanins retrieved from STRING. The data are presented as means (±SD, n = 3-5). One-way ANOVA was used to show significance and was illustrated as follows: ** p < 0.01,** * p < 0.001,**** p < 0.0001.

Article Snippet: 50 μl of purified EVs at a concentration of 1 × 10 10 /ml were stained with either anti-human CD9 (Miltenyi Biotech, clone SN4), anti-human CD63 (Miltenyi Biotec, clone H5C6) and anti-human CD81 antibodies (Beckman Coulter, clone JS64) or REA control APC conjugated antibodies at a concentration of 8 nM overnight at room temperature in the dark.

Techniques: Transduction, Expressing, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Generation of CD63-mNG-EVs in WT, PanKO-, CD9KO-, CD63KO-, and CD81KO-cells. A. Schematic workflow of engineered mNG-EVs by introducing CD63-mNG lentiviruses “created with BioRender.com ”. B. Percentage of mNG positive cells after transduction using flow cytometry. C. MFI of the cells using flow cytometry. D. The flow cytometry plot for the cells after transduction, stained with APC-conjugated CD9/CD63/CD81 tetraspanin antibodies. E. Quantification of engineered CD63-mNG EVs from 17 µL of CM collected from KO) and WT cells. F. Imaging flow cytometry plot for the mNG-EVs derived from stably expressing mNG cells. The data are presented as means (±SD, n = 2-3). One-way ANOVA was used to show significance and was illustrated as follows: * p< 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: 50 μl of purified EVs at a concentration of 1 × 10 10 /ml were stained with either anti-human CD9 (Miltenyi Biotech, clone SN4), anti-human CD63 (Miltenyi Biotec, clone H5C6) and anti-human CD81 antibodies (Beckman Coulter, clone JS64) or REA control APC conjugated antibodies at a concentration of 8 nM overnight at room temperature in the dark.

Techniques: Transduction, Flow Cytometry, Staining, Imaging, Derivative Assay, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Proteomic evaluation on the restoration and expression of CD63 in EVs. (A) Expression of CD63 in EVs originating from CD63KO and WT HEK293T cells. The graph indicates the relative difference in CD63 level across samples using log2-transformed protein intensities centered across all samples. (B) Expression of CD63 in EVs from CD63KO cells transfected with Tluc-CD63 compared to CD63KO and WT cells. The graph indicates the log2 fold change in the relative CD63 level over CD63 KO EVs or WT EVs. Results represent data from three biological replicates.

Article Snippet: 50 μl of purified EVs at a concentration of 1 × 10 10 /ml were stained with either anti-human CD9 (Miltenyi Biotech, clone SN4), anti-human CD63 (Miltenyi Biotec, clone H5C6) and anti-human CD81 antibodies (Beckman Coulter, clone JS64) or REA control APC conjugated antibodies at a concentration of 8 nM overnight at room temperature in the dark.

Techniques: Expressing, Transformation Assay, Transfection