mouse coagulation factor vii antibody  (Bio-Techne corporation)

Journal: Cancers

Article Title: Factor VIIa Regulates the Level of Cell-Surface Tissue Factor through Separate but Cooperative Mechanisms

doi: 10.3390/cancers13153718

Figure Lengend Snippet: Analysis of the association between TF, fVII and PAR2 on the surface of cells: MDA-MB-231 cells (10 3 ) were seeded out into 35 mm-glass based μ-dishes and incubated overnight. Cells were then fixed with 4% ( v / v ) paraformaldehyde for 15 min, washed three times with PBS for 5 min and blocked with Duolink blocking buffer for 1 h. The cells were then incubated overnight at 4 °C with combinations of antibodies as follows: the cells were probed with a mouse anti-fVII antibody (321621; 10 µg/mL) together with a rabbit anti-TF antibody (FL295; 5 µg/mL) to examine the interaction between fVII and TF. Another set of cells were probed with a rabbit anti-TF antibody (FL295; 5 µg/mL) and a mouse anti-PAR2 antibody (SAM11; 20 μg/mL) to examine the association of TF and PAR2 proteins. Finally, to test the proximity between fVII and PAR2, the cells were probed with a polyclonal rabbit anti-fVII antibody (10 µg/mL) together with mouse anti-PAR2 antibody (SAM11). ( A ) The dishes were washed three times with PBS and PLA performed according to the manufacturer’s instructions. The cells were stained with DAPI (2 μg/mL) and Phalloidin-FITC (2 µg/mL). Images were acquired using a Zeiss Axio Vert.A1 inverted fluorescence microscope with a ×40 magnification. A different representative of the control samples in which one primary antibody was substituted with a isotype control is presented in each of the figures. ( B ) The number of red fluorescent events and nuclei were determined as incidence/cell in 10 fields of view from each assay using the ImageJ program. ( n = 4; * = p < 0.05 vs. the control).

Article Snippet: To examine the interaction between fVII/fVIIa and TF, cells were probed with a mouse anti-fVII antibody (321621; 10 µg/mL; R&D Systems, Abingdon, UK) together with a rabbit anti-TF antibody (FL295; 5 µg/mL; Santa Cruz Biotechnologies).

Techniques: Incubation, Blocking Assay, Staining, Fluorescence, Microscopy

Journal: Cancers

Article Title: Factor VIIa Regulates the Level of Cell-Surface Tissue Factor through Separate but Cooperative Mechanisms

doi: 10.3390/cancers13153718

Figure Lengend Snippet: Analysis of the association of TF and PAR2 in the presence and absence of fVIIa: Sets of MDA-MB-231 cells (10 5 ) cells were transfected with a set of Silencer Select Pre-designed siRNA specific for the coagulation factor VII or a control siRNA (200 nM) using Trans IT-2020 transfection reagent and incubated for 48 h at 37 °C. ( A ) The knock-down was optimised beforehand by Western blot using a rabbit polyclonal anti-fVII antibody 1:2000 ( v / v ) and developed with a goat anti-rabbit antibody 1:4000 ( v / v ) (also see ). The membranes were analysed by the ImageJ program to determine the level of expression of fVII as well as the active subunits of fVIIa protein. ( n = 3; * = p < 0.05 vs. Non-transfected sample). ( B ) MDA-MB-231 cells (10 3 ) were seeded out into 35 mm-glass based μ-dishes overnight. Sets of cells were transfected and incubated as above. Some transfected cells were supplemented with purified fVIIa (10 nM) and other untransfected sets of cells were treated with apixaban (1 µg/mL). All cells were then fixed with 4% ( v / v ) paraformaldehyde for 15 min, washed three times with PBS for 5 min and blocked with Duolink blocking buffer for 1 h. The cells were then incubated overnight at 4 °C with a rabbit anti-TF antibody (FL295; 5 µg/mL) and a mouse anti-PAR2 antibody (SAM11; 20 μg/mL). The dishes were washed three times with PBS and PLA performed according to the manufacturer’s instructions. The cells were stained with DAPI (2 μg/mL) and images were acquired using a Zeiss Axio Vert.A1 inverted fluorescence microscope with a ×40 magnification. A different representative of the control samples in which one primary antibody was substituted with a isotype control, is presented in each of the figures. ( C ) The number of red fluorescent events and nuclei were determined as incidence/cell in 10 fields of view from each assay using the ImageJ program. ( n = 3; * = p < 0.05 vs. Sample containing control siRNA).

Article Snippet: To examine the interaction between fVII/fVIIa and TF, cells were probed with a mouse anti-fVII antibody (321621; 10 µg/mL; R&D Systems, Abingdon, UK) together with a rabbit anti-TF antibody (FL295; 5 µg/mL; Santa Cruz Biotechnologies).

Techniques: Transfection, Coagulation, Incubation, Western Blot, Expressing, Purification, Blocking Assay, Staining, Fluorescence, Microscopy

Journal: Cancers

Article Title: Factor VIIa Regulates the Level of Cell-Surface Tissue Factor through Separate but Cooperative Mechanisms

doi: 10.3390/cancers13153718

Figure Lengend Snippet: Analysis of the association of recombinant fVIIa with caveolin-1 on the surface of HDBEC on addition of recombinant TF: HDBEC (10 3 ). were seeded out into 35 mm-glass based μ-dishes overnight and treated with recombinant TF (1–4 U/mL) for 10 or 40 min. Cells were then fixed with 4% ( v / v ) paraformaldehyde for 15 min, washed three times with PBS for 5 min and blocked with Duolink blocking buffer for 1 h. The cells were then incubated overnight at 4 °C with a mouse anti-fVII antibody (321621; 10 µg/mL) and a polyclonal rabbit anti-caveolin-1 antibody (10 μg/mL). ( A ) The dishes were washed three times with PBS and PLA performed according to the manufacturer’s instructions. The cells were stained with DAPI (2 μg/mL) and images were acquired using a Zeiss Axio Vert.A1 inverted fluorescence microscope with a ×40 magnification. ( B ) The number of red fluorescent events and nuclei were determined as incidence/cell in 10 fields of view from each assay using the ImageJ program. ( n = 3; * = p < 0.05 vs. No fVII antibody; # = p < 0.05 vs. No TF sample).

Article Snippet: To examine the interaction between fVII/fVIIa and TF, cells were probed with a mouse anti-fVII antibody (321621; 10 µg/mL; R&D Systems, Abingdon, UK) together with a rabbit anti-TF antibody (FL295; 5 µg/mL; Santa Cruz Biotechnologies).

Techniques: Recombinant, Blocking Assay, Incubation, Staining, Fluorescence, Microscopy

Journal: Oncotarget

Article Title: Chimeric antigen receptor-modified T Cells inhibit the growth and metastases of established tissue factor-positive tumors in NOG mice

doi: 10.18632/oncotarget.14367

Figure Lengend Snippet: ( A ) Structure diagram of TF-CAR. mlF VII, the light chain of mouse coagulation factor VII; TMD, transmembrane domain. ( B ) Western bloting analysis of TF-CAR expression in T cells. CAR-T, TF-CAR T cells; CON-T, vector-transduced T cells; T, untreated T cells. ( C ) Flow cytometry analysis of TF-CAR expression in transduced T cells. ( D ) Immunofluorescence staining analysis of TF-CAR expression in transduced T cells. The images were taken under × 400 magnification. ( E ) CAR positive ratio analysis of lentivirus infected T cells by Flow cytometry. All sample were prepared after 5 days of virus infection.

Article Snippet: The following antigens and antibodies were used for this analysis: human TF, mouse anti-human TF primary antibody (R&D Systems, Minnesota, USA) and FITC-conjugated anti-mouse IgG1 secondary antibody (eBioscience, San Diego, CA); mouse TF, PE-conjugated goat anti-mouse TF antibody (R&D Systems, Minnesota, USA); TF-CAR, polyclonal goat anti-mouse coagulation factor VII primary antibody (R&D Systems, Minnesota, USA) and PE-conjugated rabbit anti-goat IgG secondary antibody (Santa Cruz, CA, USA).

Techniques: Coagulation, Western Blot, Expressing, Plasmid Preparation, Flow Cytometry, Immunofluorescence, Staining, Infection, Virus