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Bd Biosciences - Discovery Labware Bd Biocoat angiogenesis system: endothelial cell invasion • Evaluate endothelial cell invasion using real-time fluorescence detection in a simplified and reproducible manner • Increase screening throughput for prospective pro- and antiangiogenic compounds During angiogenesis, endothelial cells are activated and express matrix metalloproteinases (MMPs), which degrade the vascular basement membrane and facilitate cellular movement towards angiogenic stimuli 1,2 .The BD BioCoat Angiogenesis System: Endothelial Cell Invasion provides an in vitro, quantitative assay for prospective anti- and pro-angiogenic compounds. It is composed of a BD Falcon 24-Multiwell Insert Plate (and a non TC-treated 24- or 96-well receiver plate and lid) containing a BD FluoroBlok fluorescence-blocking microporous (3.0 µm pore size) polyethylene terephthalate (PET) membrane coated with BD Matrigel Matrix. BD Matrigel Matrix is a reconstituted basement membrane preparation derived from Engelbreth-Holm-Swarm (EHS) mouse tumors. Furthermore, this matrix is primarily composed of laminin, collagen IV, entactin, and a number of growth factors. An optimized coating process effectively occludes the membrane pores and provides a functional barrier that is analogous to the basement membrane in vivo. This coating blocks the passage of non-invasive cells and allows the passage of activated endothelial cells with invasive capacity. Following activation by angiogenic factors, the endothelial cells express MMP 2 and 9. These proteases digest the matrix and enable the cells to invade through the matrix barrier to the bottom side of the microporous membrane 1,3 . Unlike traditional in vitro cell invasion assays, the BD Endothelial Cell Invasion Assay allows for rapid data collection without multiple handling steps (i.e., plate washing, manual cell scraping, and manual counting). Since the BD FluoroBlok membrane effectively blocks the fluorescence of labeled cells that have not invaded through the membrane, only cells that appear on the underside of the BD FluoroBlok membrane are detected. Cells may be labeled with a fluorescent dye either pre- or post-invasion. The BD FluoroBlok membrane effectively blocks > 99% of the excitation and emission wavelengths of fluorophores commonly used to label cells (see Figure 1). This reproducible system routinely yields inter- and intraassay CVs of ≤ 15%. This assay has been shown to be suitable for use with human microvascular endothelial cells (HMVEC) and the human microvascular endothelial cell line, HMEC-1. Using this system, endothelial cell invasion occurs in response to angiogenic factors such as VEGF and bFGF. Furthermore, the inhibition of endothelial cell invasion has been demonstrated using known antiangiogenic agents. Hmvec invasion through Bd Biocoat angiogenesis system: endothelial cell invasion - Serum + 5% Serum Human microvascular endothelial cells (HMVECs) were placed in the BD BioCoat Angiogenesis System: Endothelial Cell Invasion in either the absence (control) or presence of serum and allowed to invade for 22±1 hour . Cells were labeled post-invasion with Calcein AM and visualized using an Olympus IMT-2 phase epifluorescent microscope . Images were captured using IPWIN 4 .0 software . REFERENCES: 1. Harris, S.R., et al., In Vivo, 12:563 (1998). 2. Molema, G., Pharmacological Reviews, 52(2):237 (2000). 3. Benelli, R., et al., The Int. J. Biol. Markers, 14(4):243 (1999). Fluorescent Units 1800 1600 1400 1200 1000 800 600 400 200 0 Control drug discovery and development VEGF (4 ng/ml) 0.1 µg/ml 1 µg/ml 10 µg/ml 20 µg/ml VEGF (4 ng/ml) + 1'10' Phenanthroline Effect of MMP Inhibitor 1’10’ Phenanthroline on HMVEC Invasion HMVECs were assayed in the BD BioCoat Angiogenesis System: Endothelial Cell Invasion in the absence (Control) or presence of VEGF (4 ng/ml) with varying concentrations of 1’10’ Phenanthroline in the bottom chamber . Cells were allowed to invade for 22±1 hour . Cells were labeled post-invasion with Calcein AM (4 µg/ml) and measured by detecting the fluorescence of cells that invaded through the BD Matrigel Matrix with an Applied Biosystems CytoFluor® 4000 plate reader at 485 nm excitation and 530 nm emission . Data represents the mean of n=3 inserts ± SD . Quality control: • Tested for its ability to allow invasion of HMVEC-1 cells, an invasive human microvascular endothelial cell line, and to exclude invasion of NIH-3T3 cells, a non-invasive fibroblast cell line • Tested and found negative for bacteria and fungi storage and stability: Stable for at least three months from the date of shipping when stored at -20°C. TIP description Qty. cat. no. Bd Biocoat angiogenesis system: endothelial cell invasion 24-Multiwell Insert System 1 354141 24-Multiwell Insert System 5 354142 Remember to order BD Falcon FluoroBlok 24-Multiwell Insert System as control plates when purchasing the BD BioCoat Angiogenesis System: Endothelial Cell Invasion . RELATED PRODUCTS More BD BioCoat Angiogenesis Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . .next pages BD BioCoat Endothelial Cell Growth Environment . . . . . . . . . . . . . . . . . . . 68 BD Vascular Endothelial Growth Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . 194 7 insert systems bdbiosciences.com | 149
7 insert systems drug discovery and development Bd Biosciences - Discovery Labware Bd Biocoat angiogenesis system: endothelial cell migration • A quantitative and reproducible in vitro model system for examining the effects of prospective compounds on endothelial cell migration Cell migration (or chemotaxis) is the directional movement of cells in response to a concentration gradient of a soluble chemoattractant. During angiogenesis, activated endothelial cells invade through the basement membrane and migrate towards a variety of pro-angiogenic factors 1 . The BD BioCoat Angiogenesis System: Endothelial Cell Migration is composed of a BD Falcon 24- or 96-Multiwell Insert Plate (and a non-TC treated 24- or 96-well receiver plate and lid) containing a BD FluoroBlok fluorescence-blocking microporous polyethylene terephthalate (PET) membrane (3.0 µm pore size) evenly coated with human fibronectin. An optimized coating process is used to ensure that the pores of the membrane are not occluded. Therefore, endothelial cells attach to the coated membrane and freely migrate through the pores towards an appropriate chemoattractant in the lower chamber of the plate. To measure cell migration, a bottom-reading fluorometer is used to quantitate the number of cells that have migrated through the pores and attached to the underside of the insert membrane 2 . The cells may be labeled with a fluorescent dye either pre- or post-migration. The pre-labeling technique enables real-time kinetic measurements of cell migration. Studies conducted using the post-labeling technique demonstrated that endothelial cells migrate towards VEGF in a concentration-dependent manner. Similar results were obtained when bFGF was used as a chemoattractant. Moreover, the BD BioCoat Angiogenesis System: Endothelial Cell Migration has been used in conjunction with the Cellomics HSC ArrayScan to examine endothelial cell migration in a quantitative high-throughput assay 3 . 150 | bdbiosciences.com Fluorescent Units 6000 5000 4000 3000 2000 1000 0 Fibronectin Coated Inserts Uncoated Inserts 0 HUVEC Migration on Uncoated and HFN-Coated Inserts Migration assays were conducted using HUVECs in the BD BioCoat Angiogenesis System: Endothelial Cell Migration and compared with uncoated BD FluoroBlok 24-Multiwell Inserts (Cat . No . 351155) using both FBS (5%) and VEGF (10 ng/ml) as chemoattractants . The cells were allowed to migrate for 22±1 hour . Cells were labeled post-migration with Calcein AM (4 µg/ml) and measured by detecting the fluorescence of the cells that migrated through the fibronectin-coated BD FluoroBlok membrane using an Applied Biosystems CytoFluor® 4000 plate reader [485/530 nm (Ex/Em) wavelengths] . The data showed a significant increase in migration in response to VEGF when the assay was performed on the fibronectin-coated inserts included in the system . Data represents the mean of n=3 inserts ± S .D . VEGF REFERENCES: 1. Harris, S.R., et al., In Vivo, 12:563 (1998). 2. Goldberger, A. and Septak, M., BD Biosciences Discovery Labware, Technical Bulletin #428, (1998). 3. Mastyugin, V., et al., J. Biomol Screening, Vol. 9:712 (2004). FBS
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204 Bioimaging SyStemS BD Bioscienc
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9 Pathway Bioimager Bioimaging SySt
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9 aCtone gPCR Screening assay Bioim
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10 Alphabetical Index AlphAbetIcAl
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10 numerical Index nUMeRIcAl Index
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BD Biosciences Worldwide Discovery
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