human anti cd47 apc conjugated antibody  (R&D Systems)

Journal: Cells

Article Title: CD47 in Osteosarcoma: Correlation with Metastasis and Macrophage-Mediated Phagocytosis

doi: 10.3390/cells13221862

Figure Lengend Snippet: Characteristics of the 24 patients treated at the National Cancer Center Hospital between 2003 and 2021.

Article Snippet: Immunohistochemical staining was performed using allophycocyanin (APC)-conjugated anti-human CD47 (B6H12; Thermo Fisher Scientific, Waltham, MA, USA) and APC-conjugated mouse IgG1κ isotype control (MOPC-21; BioLegend, San Diego, CA, USA) antibodies.

Techniques:

Journal: Cells

Article Title: CD47 in Osteosarcoma: Correlation with Metastasis and Macrophage-Mediated Phagocytosis

doi: 10.3390/cells13221862

Figure Lengend Snippet: Immunohistochemical expression of CD47 in biopsy samples. The depicted regions were selected from incisional biopsy samples, and immunohistochemical staining was performed as described under Materials and Methods. The upper panel (A, from case 14) depicts CD47-negative expression, while the lower panel (B, from case 15) depicts CD47-positive expression, showing CD47-positive cells stained in light brown color.

Article Snippet: Immunohistochemical staining was performed using allophycocyanin (APC)-conjugated anti-human CD47 (B6H12; Thermo Fisher Scientific, Waltham, MA, USA) and APC-conjugated mouse IgG1κ isotype control (MOPC-21; BioLegend, San Diego, CA, USA) antibodies.

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: Cells

Article Title: CD47 in Osteosarcoma: Correlation with Metastasis and Macrophage-Mediated Phagocytosis

doi: 10.3390/cells13221862

Figure Lengend Snippet: Comparison of clinicopathological characteristics based on CD47 positivity.

Article Snippet: Immunohistochemical staining was performed using allophycocyanin (APC)-conjugated anti-human CD47 (B6H12; Thermo Fisher Scientific, Waltham, MA, USA) and APC-conjugated mouse IgG1κ isotype control (MOPC-21; BioLegend, San Diego, CA, USA) antibodies.

Techniques: Comparison, Biomarker Discovery

Journal: Cells

Article Title: CD47 in Osteosarcoma: Correlation with Metastasis and Macrophage-Mediated Phagocytosis

doi: 10.3390/cells13221862

Figure Lengend Snippet: In vitro efficacy of CD47 antibody in osteosarcoma cells. ( A ) The expression of CD47 is analyzed using FACS analysis in osteosarcoma cells. Representative images display the histogram of CD47 expression under basal conditions. ( B – D ) Osteosarcoma cells are treated with either isotype control or anti-CD47 (B6H12) antibody at the indicated concentrations for 24, 48, and 72 h. The effect of the anti-CD47 antibody on osteosarcoma cell viability is assessed using the EZ-CyTox cell viability assay kit. ( E ) KHOS/NP osteosarcoma cells are exposed to an isotype control or anti-CD47 (B6H12) antibody at the indicated concentrations for 24, 48, and 72 h. The effect of the anti-CD47 antibody on KHOS/NP cell apoptosis is evaluated using the Annexin V Detection kit.

Article Snippet: Immunohistochemical staining was performed using allophycocyanin (APC)-conjugated anti-human CD47 (B6H12; Thermo Fisher Scientific, Waltham, MA, USA) and APC-conjugated mouse IgG1κ isotype control (MOPC-21; BioLegend, San Diego, CA, USA) antibodies.

Techniques: In Vitro, Expressing, Control, Viability Assay

Journal: Cells

Article Title: CD47 in Osteosarcoma: Correlation with Metastasis and Macrophage-Mediated Phagocytosis

doi: 10.3390/cells13221862

Figure Lengend Snippet: Wound-healing assay to assess migration of osteosarcoma cells following CD47 (B6H12) antibody treatment. ( A ) MG-63 osteosarcoma cells are treated with a 10 μg/mL concentration of an isotype control antibody or the anti-CD47 antibody (B6H12). Representative images show the area the cells cover at the indicated time points after in vitro scratch wounding. ( B ) The summary graph illustrates the percentage of wound closure in MG-63 osteosarcoma cells over time compared with untreated controls. Statistical analysis of wound closure is conducted using ImageJ software with the wound healing size plug-in. The percentages are represented as the mean ± standard error of the mean, and statistical significance is determined by comparison with the control (* p < 0.05).

Article Snippet: Immunohistochemical staining was performed using allophycocyanin (APC)-conjugated anti-human CD47 (B6H12; Thermo Fisher Scientific, Waltham, MA, USA) and APC-conjugated mouse IgG1κ isotype control (MOPC-21; BioLegend, San Diego, CA, USA) antibodies.

Techniques: Wound Healing Assay, Migration, Concentration Assay, Control, In Vitro, Software, Comparison

Journal: Cells

Article Title: CD47 in Osteosarcoma: Correlation with Metastasis and Macrophage-Mediated Phagocytosis

doi: 10.3390/cells13221862

Figure Lengend Snippet: Wound-healing assay to assess migration of osteosarcoma cells following CD47 siRNA transfection. ( A ) MG-63 osteosarcoma cells are treated with either control siRNA or CD47 siRNA, and Western blot analysis is performed to evaluate CD47 expression. The summary graph illustrates the CD47 expression levels in the MG-63 osteosarcoma cells. ( B ) Representative images display the area covered by MG-63 osteosarcoma cells at the indicated time points following in vitro scratch wounding. The summary graph illustrates the percentage of wound closure in MG-63 cells over time compared to the control siRNA. Statistical analysis of wound closure is performed using ImageJ software with the wound healing size plug-in. The percentages are represented as the mean ± standard error of the mean, and statistical significance is determined by comparison with the control siRNA (** p < 0.01, n.s. : no significance).

Article Snippet: Immunohistochemical staining was performed using allophycocyanin (APC)-conjugated anti-human CD47 (B6H12; Thermo Fisher Scientific, Waltham, MA, USA) and APC-conjugated mouse IgG1κ isotype control (MOPC-21; BioLegend, San Diego, CA, USA) antibodies.

Techniques: Wound Healing Assay, Migration, Transfection, Control, Western Blot, Expressing, In Vitro, Software, Comparison

Journal: Cells

Article Title: CD47 in Osteosarcoma: Correlation with Metastasis and Macrophage-Mediated Phagocytosis

doi: 10.3390/cells13221862

Figure Lengend Snippet: Phenotypic characterization of monocyte-derived macrophages and comparative analysis of in vitro macrophage-mediated phagocytosis following CD47 (B6-H12) antibody treatment. ( A ) CD14-positive monocytes are isolated and cultured in a macrophage medium, followed by activation with recombinant human M-CSF for 6 d. Single cells are stained and analyzed using the BD FACSVerse System. ( B , C ) KHOS/NP cells are labeled with CellTrace™ CFSE dye (KHOS-CFSE) and incubated with 10 μg/mL anti-CD47 antibody or IgG ISO-type antibody for 30 min at 37 °C. KHOS/NP-CFSE cells are co-cultured with differentiated macrophages for 2 h. The percentage of macrophage-mediated phagocytosis (CD14-high, CellTrace-high, and CD11b-high) is measured by flow cytometry. Data are recorded using BD FACSVerse and analyzed using FlowJo software. The percentages are presented as the mean ± standard error of the mean, and statistical significance is determined by comparison with the IgG ISO-type antibody (** p < 0.01).

Article Snippet: Immunohistochemical staining was performed using allophycocyanin (APC)-conjugated anti-human CD47 (B6H12; Thermo Fisher Scientific, Waltham, MA, USA) and APC-conjugated mouse IgG1κ isotype control (MOPC-21; BioLegend, San Diego, CA, USA) antibodies.

Techniques: Derivative Assay, In Vitro, Isolation, Cell Culture, Activation Assay, Recombinant, Staining, Labeling, Incubation, Flow Cytometry, Software, Comparison

Journal: Journal of nanobiotechnology

Article Title: Thrombolytic therapy based on lyophilized platelet-derived nanocarriers for ischemic stroke.

doi: 10.1186/s12951-023-02206-5

Figure Lengend Snippet: Fig. 1 A Schematic representation of the preparation of rtPA-loaded CSM derived from platelets. B Representative mean hydrodynamic diameter of CSM and CSM@rtPA before and after lyophilization (CSM@rtPA/L) process measured by DLS. Data represent mean ± SEM (n = 3, independent samples). C Representative mean diameter of CSM and CSM@rtPA before and after lyophilization process (CSM@rtPA/L) measured by NTA. Data represent mean ± SEM (n = 3, independent samples). D Representative STEM-in SEM images of CSM@rtPA negatively stained with uranyl acetate. Scale bars: 200 nm. E Loading capacity of rtPA encapsulated in CSM samples. Data represent mean ± SEM (n = 3, independent samples). F Representative density plots and quantitative analysis of CSM and CSM@rtPA measured by FC. Scatter density plots of Green fluorescence signal (Green-B channel, rtPA channel) versus Red fluorescence signal (Red-R channel, CellMask DeepRed channel) for CSM, CSM@rtPA, and CSM@rtPA sample after labeling with CellMask Deep Red for lipid staining. Mean fluorescence intensities (MFI) for Green-B channel (D) and Red-R channel. Data represent mean ± SEM (n = 3, independent samples). G. Schematic representation of platelets and platelet-derived CSM surface proteins studied by APC-fluorescently labeled antibodies: anti-hCD47 Ab and anti-hCD42b/GPlba Ab. In vitro Ab binding to platelets and CSM@rtPA before and after lyophilization process. Representative MFI histogram of Red-R channel (APC signal) for platelets, CSM@rtPA and CSM@rtPA/L samples after incubation with APC-anti-CD47 and APC-anti-CD42b/GPIbα antibodies

Article Snippet: Then, fluorescently antibodies, human anti-CD47 APC conjugated Antibody (FAB4670A, R&D Systems) and human antiCD42b/GPlbα APC conjugated Antibody (FAB4067A, R&D systems) were incubated with platelets and with CSMs (10 μL of antibody stock solution /106 cells) and the samples were analyzed by flow cytometer.

Techniques: Derivative Assay, Lyophilization, Staining, Fluorescence, Labeling, In Vitro, Binding Assay, Incubation

Journal: bioRxiv

Article Title: HIV-1 Vpu promotes phagocytosis of infected CD4 + T cells by macrophages through downregulation of CD47

doi: 10.1101/2021.03.16.435750

Figure Lengend Snippet: SupT1 T cells or primary CD4 + T cells were infected with GFP-expressing NL4-3 (WT or dU) viruses or with either VSV-G pseudotyped GFP-expressing NL 4-3 ADA (WT or dU) or transmitted/founder WITO (WT or dU) viruses as indicated. After 48 h, cells were stained with anti-CD47 (clone CC2C6) and analyzed by flow cytometry. (Top) Representative flow cytometry dot-plot graphs with indication of the median fluorescence intensity (MFI) values for infected (GFP- or Gag-positive) and bystander cells (GFP- or Gag-negative). (Bottom) Summary graphs of relative surface CD47 expression levels at 48 h postinfection (hpi) with the indicated viruses (n=4). The percent MFI values were calculated relative to that obtained in the respective bystander cells. Statistical analysis was performed using Mann-Whitney U-test (**, P < 0.01; *, P < 0.05), error bars represent standard deviations (SD). Flow cytometry data for this figure was generated on a CyAn ADP cytometer (Beckman coulter).

Article Snippet: For flow cytometry, the following antibodies (Abs) were used: PE/Cy7-conjugated mouse anti-human CD47 (clone CC2C6) monoclonal Ab (mAb) and APC-conjugated anti-CD11b mAb (clone ICRF44) as well as corresponding isotype controls from BioLegend, APC-conjugated mouse anti-human CD47 mAb (clone B6H12; eBioscience), RD1-conjugated anti-Gag (clone KC57; Beckman Coulter).

Techniques: Infection, Expressing, Staining, Flow Cytometry, Fluorescence, MANN-WHITNEY, Generated, Cytometry

Journal: bioRxiv

Article Title: HIV-1 Vpu promotes phagocytosis of infected CD4 + T cells by macrophages through downregulation of CD47

doi: 10.1101/2021.03.16.435750

Figure Lengend Snippet: (A) Experimental strategy for HIV-1-infected target cells labelling, coculture with MDMs for analysis of in vitro capture or phagocytosis by flow cytometry. Target cells were either mock-infected or infected with VSV-G pseudotyped NL 4-3 ADA (WT or dU) viruses for 48 h and labelled with either CFSE (left) or pHrodo (right). (B) Representative flow cytometry dot-plots of MDMs (CD11b + ) with percentage numbers of CFSE + population corresponding to capture (top); summary graphs for capture of CFSE-labelled CD47 expressing Jurkat E6.1 control (ctrl) or CD47 knockout (KO) Jurkat E6.1 cells by MDMs in the indicated conditions (bottom). (C) Representative flow cytometry dot-plots of MDMs (CD11b + ) with percentage numbers of pHrodo + populations corresponding to phagocytosis (top); summary graphs for phagocytosis of pHrodo-labelled target cells by MDMs in the indicated conditions (bottom). (B and C) analyzed by Mann-Whitney U-test (*, P < 0.05; ns, nonsignificant, P > 0.05), error bars represent SD.

Article Snippet: For flow cytometry, the following antibodies (Abs) were used: PE/Cy7-conjugated mouse anti-human CD47 (clone CC2C6) monoclonal Ab (mAb) and APC-conjugated anti-CD11b mAb (clone ICRF44) as well as corresponding isotype controls from BioLegend, APC-conjugated mouse anti-human CD47 mAb (clone B6H12; eBioscience), RD1-conjugated anti-Gag (clone KC57; Beckman Coulter).

Techniques: Infection, In Vitro, Flow Cytometry, Expressing, Knock-Out, MANN-WHITNEY

Journal: bioRxiv

Article Title: HIV-1 Vpu promotes phagocytosis of infected CD4 + T cells by macrophages through downregulation of CD47

doi: 10.1101/2021.03.16.435750

Figure Lengend Snippet: (A) Vpu induces depletion of CD47. HEK 293T cells were cotransfected with plasmids encoding HA-tagged CD47 (pCD47-HA), along with increasing concentrations of GFP-marked plasmids expressing wild-type ADA Vpu (pVpu). An empty vector expressing GFP alone was added to adjust the total amounts of plasmid DNAs in all conditions. Whole cell lysates were analyzed for the indicated proteins by Western blotting. A Representative blot is shown (top), and a summary graph of densitometric analysis of CD47 is presented (bottom), error bars represent SD. (B) Vpu-mediated CD47 depletion requires the main Vpu functional motifs. HEK 293T were cotransfected with pCD47-HA, along with either empty vector, or plasmids encoding WT Vpu, or the indicated Vpu mutants. A representative Western blot is shown (top) as well as a summary graph of densitometric analysis of CD47 (bottom); statistical significance was determined by Mann-Whitney U-test (*, P < 0.05; ns, nonsignificant, P > 0.05); error bars represent SD. (C) HEK 293T cells were co-transfected with the indicated plasmids for 48 h prior to cell lysis and immunoprecipitation (IP) using anti-HA antibody. The immunoprecipitates were analyzed for the indicated proteins by Western blotting. (D) HEK 293T cells were cotransfected with the indicated plasmids for 36 h and vehicle (DMSO), MG132 or Concanamycin A (ConA) were added 8 h before cells were harvested and analyzed by Western blotting.

Article Snippet: For flow cytometry, the following antibodies (Abs) were used: PE/Cy7-conjugated mouse anti-human CD47 (clone CC2C6) monoclonal Ab (mAb) and APC-conjugated anti-CD11b mAb (clone ICRF44) as well as corresponding isotype controls from BioLegend, APC-conjugated mouse anti-human CD47 mAb (clone B6H12; eBioscience), RD1-conjugated anti-Gag (clone KC57; Beckman Coulter).

Techniques: Expressing, Plasmid Preparation, Western Blot, Functional Assay, MANN-WHITNEY, Transfection, Lysis, Immunoprecipitation

Journal: bioRxiv

Article Title: HIV-1 Vpu promotes phagocytosis of infected CD4 + T cells by macrophages through downregulation of CD47

doi: 10.1101/2021.03.16.435750

Figure Lengend Snippet: (A) Flow cytometry histogram to validate CD47 surface expression levels in different target Jurkat cell lines including JC47 (CD47 knockout), JC47-hCD47 (human CD47 reintroduced in JC47), JC47-cCD47 (chimeric CD47 reintroduced in JC47). (B) JC47-hCD47 and JC47-cCD47 cells were mock-infected or infected with VSV-G pseudotyped GFP-expressing WT NL 4-3 ADA virus for 48 h, then stained with anti-CD47mAb (clone CC2C6) and analysed by flow cytometry. Representative flow cytometry dot-plot graphs with the MFI values in infected (GFP-positive) and bystander cells (GFP-negative), (left); summary graphs of relative surface CD47 expression levels at 48 h after infection (n=4), (right), the percent MFI values were calculated relative to respective their GFP-negative cells. Statistical analysis was performed using Mann-Whitney U-test (*, P < 0.05); error bars represent SD. (C) The indicated mock or HIV-1-infected target cells were labelled with pHrodo and cocultured with MDMs for 2 h, prior to analysis of MDMs by flow cytometry. Representative flow cytometry dot-plots of MDMs (CD11b + ) with percentage of pHrodo + populations corresponding to phagocytosis of target cells by MDMs (left); summary graphs for MDMs (right) from 6 distinct donors, analyzed by Wilcoxon matched-pairs signed rank test (*, P < 0.05; ns, nonsignificant, P > 0.05). (D) JC47-hCD47 or JC47-cCD47 cells were infected as described and cocultured with MDMs for 6 h. Following washing-off of infected T cells, MDMs were cultured for 2 days, media of MDMs was collected to infect TZM-bl, for luciferase assay. Shown are RLU detected with media collected from MDMs of 3 distinct donors, error bars represent SD.

Article Snippet: For flow cytometry, the following antibodies (Abs) were used: PE/Cy7-conjugated mouse anti-human CD47 (clone CC2C6) monoclonal Ab (mAb) and APC-conjugated anti-CD11b mAb (clone ICRF44) as well as corresponding isotype controls from BioLegend, APC-conjugated mouse anti-human CD47 mAb (clone B6H12; eBioscience), RD1-conjugated anti-Gag (clone KC57; Beckman Coulter).

Techniques: Flow Cytometry, Expressing, Knock-Out, Infection, Staining, MANN-WHITNEY, Cell Culture, Luciferase