polyclonal biotinylated antibodies against vegf 165  (R&D Systems)

Journal: PLoS ONE

Article Title: The Role of Vascular Actors in Two Dimensional Dialogue of Human Bone Marrow Stromal Cell and Endothelial Cell for Inducing Self-Assembled Network

doi: 10.1371/journal.pone.0016767

Figure Lengend Snippet: (A) Gene expression VEGF 165 in HBMSC, HUVEC, co-HBMSC and co-HUVEC after being cultured for different time. Data of gene expression was quantified relative to the same gene expression in HBMSC after 6 hours of culture (same in the following gene expression analysis). a and b indicated the difference p≤0.05 or p≤0.01, respectively. Interestingly, we found that VEGF 165 was only expressed by HBMSC and co-HBMSC and VEGF 165 expression in co-HBMSC is much higher than that in HBMSC during 6 hours–18 hours. However, this upregulation in co-HBMSC diminished at 24 hours. (B) Gene expression of KDR in HBMSC, HUVEC, co-HBMSC and co-HUVEC after being cultured for different time. KDR was mainly expressed by endothelial cells and it was much higher in co-HUVEC than in HUVEC from 14–24 hours. At 6 hours, no significant difference has been observed. (C) Gene expression of uPA in HBMSC, HUVEC, co-HBMSC and co-HUVEC after being cultured for different time. uPA has a similar expression style as to the expression of KDR. (D) Gene expression of uPAR in HBMSC, HUVEC, co-HBMSC and co-HUVEC after being cultured for different time. uPAR expressed by all cells and it is higher expressed by co-cultured cells as compared to mono-cultured cell. (E) Immunofluorescence staining of KDR in cells in green. Nuclear was stained in blue with DAPI. Scare bars represent 50 µm. It can be clearly seen that the expression of KDR is much higher in co-cultured cells than in mono-cultured HUVEC. No much KDR could be detected in HBMSC by immunofluorescence.

Article Snippet: Mouse monoclonal antibody against human VE-cad (Hycult Biotechnology) was used at 15 µg/ml, rabbit polyclonal antibody against human VEGF 165 (Millpore) was used at 10 µg/ml and rabbit polyclonal antibody against human uPAR (American Diagostica Inc.) was used at 5 µg/ml.

Techniques: Expressing, Cell Culture, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: The Role of Vascular Actors in Two Dimensional Dialogue of Human Bone Marrow Stromal Cell and Endothelial Cell for Inducing Self-Assembled Network

doi: 10.1371/journal.pone.0016767

Figure Lengend Snippet: (A) HBMSC and HUVEC were co-cultured with neutralizing antibodies against VE-cad, VEGF 165 and uPAR for 24 hours. We found that the neutralization of VE-cad, VEGF 165 and uPAR suppressed the formation of self-assembled network in co-culture system without impairing the viability of cells. Addition of isotype control mouse antibody or normal IgG rabbit antibody had no effect on self-assembled network formation. Scare bars represent 200 µm. (B) The migration velocity of co-HUVEC were analyzed from the images taken by time-lapse videomicroscopy observing co-cultures with or without neutralizing antibody. Interestingly, co-HUVEC in co-culture incubated with VE-cad neutralizing antibody was still be able to migrate. The migration speed is very close to the co-HUVEC in co-culture without any neutralizing antibody. However, the neutralization of VEGF 165 and uPAR totally blocked the migration of co-HUVEC in co-culture system.

Article Snippet: Mouse monoclonal antibody against human VE-cad (Hycult Biotechnology) was used at 15 µg/ml, rabbit polyclonal antibody against human VEGF 165 (Millpore) was used at 10 µg/ml and rabbit polyclonal antibody against human uPAR (American Diagostica Inc.) was used at 5 µg/ml.

Techniques: Cell Culture, Neutralization, Co-Culture Assay, Migration, Incubation

Journal: PLoS ONE

Article Title: The Role of Vascular Actors in Two Dimensional Dialogue of Human Bone Marrow Stromal Cell and Endothelial Cell for Inducing Self-Assembled Network

doi: 10.1371/journal.pone.0016767

Figure Lengend Snippet: (A) Gene expressions of VEGF 165 in HBMSC, HUVEC, Co-HBMSC and co-HUVEC cultured with (bars with shadow) or without VE-cad neutralizing antibody. Neutralization of VE-cad has no strong effects on expression of VEGF 165 . (B) Gene expressions of uPA in HBMSC, HUVEC, Co-HBMSC and co-HUVEC cultured with (bars with shadow) or without VE-cad neutralizing antibody. Neutralization of VE-cad significantly downregulated the expression of uPA in co-HUVEC at 14 hours and 16 hours and this effect decreased at 24 hours. However, the expression of uPA in co-HUVEC still maintained a high level. (C) Gene expressions of uPAR in HBMSC, HUVEC, Co-HBMSC and co-HUVEC cultured with (bars with shadow) or without VE-cad neutralizing antibody. uPAR was transiently affected by neutralization of VE-cad. Its expression statistically decreased in co-HBMSC at 6 hours and 14 hours. (D) Gene expression of uPA in HBMSC, HUVEC, Co-HBMSC and co-HUVEC with (bars with shadow) or without VEGF 165 neutralizing antibody. Neutralization of VEGF 165 strongly suppressed the expression of uPA in co-HUVEC. (E) Gene expression of uPAR in HBMSC, HUVEC, Co-HBMSC and co-HUVEC with (bars with shadow) or without VEGF 165 neutralizing antibody. Gene expression of uPAR in co-cultured cells was downregulated all the time after the addition of VEGF 165 neutralizing antibody. a and b indicated the difference p≤0.05 or p≤0.01, respectively.

Article Snippet: Mouse monoclonal antibody against human VE-cad (Hycult Biotechnology) was used at 15 µg/ml, rabbit polyclonal antibody against human VEGF 165 (Millpore) was used at 10 µg/ml and rabbit polyclonal antibody against human uPAR (American Diagostica Inc.) was used at 5 µg/ml.

Techniques: Cell Culture, Neutralization, Expressing

Journal: PLoS ONE

Article Title: The Role of Vascular Actors in Two Dimensional Dialogue of Human Bone Marrow Stromal Cell and Endothelial Cell for Inducing Self-Assembled Network

doi: 10.1371/journal.pone.0016767

Figure Lengend Snippet: Primer sequences used in Q-PCR.

Article Snippet: Mouse monoclonal antibody against human VE-cad (Hycult Biotechnology) was used at 15 µg/ml, rabbit polyclonal antibody against human VEGF 165 (Millpore) was used at 10 µg/ml and rabbit polyclonal antibody against human uPAR (American Diagostica Inc.) was used at 5 µg/ml.

Techniques: