BD BioCoat Angiogenesis Systems

BD BioCoat™ Angiogenesis Systems

bdbiosciences.comUnless otherwise specified, all products are for research use only. Not intended for use in diagnostic or therapeutic procedures.

All applications are either tested in-house or reported in the literature. See Technical Data Sheets for details.�

A ngiogenesis is the development of

new blood vessels from pre-existing

vessels. This process is essential for

normal growth and homeostasis. However,

angiogenesis becomes altered during certain

disease states, which results in excessive or

insufficient blood vessel formation. Diseases

such as cancer, diabetic retinopathy, and

rheumatoid arthritis are characterized

by excessive angiogenesis. A variety of

conditions are associated with insufficient

angiogenesis and reduced blood flow (e.g.,

stroke, coronary artery disease, and delayed

wound healing). In an attempt to restore

normal blood flow, these disorders must be

targeted with therapeutic strategies that

effectively reduce or enhance angiogenesis.

Various in vitro assays have been developed to examine the mechanisms underlying angiogenesis and to identify potential therapeutic molecules.1,� These assays address key aspects of the angiogenesis process, such as endothelial cell adhesion, proliferation, permeabilization, migration, invasion, tubulogenesis, and vessel stabilization. However, it is recognized that many of these assays are cumbersome, laborious, difficult to quantitate, and not

standardized.

The lack of effective tools for studying angiogenesis has constrained efforts to identify therapeutic compounds that

inhibit one or more components of the angiogenesis process. The BD BioCoat™ Angiogenesis Systems facilitate investigation of compound effects on endothelial cell invasion, migration, and tube formation. The availability of these standardized assays has facilitated a better understanding of the molecular mechanisms that govern angiogenesis and has simplified the routine use of cell-based assays for screening of anti- and pro-angiogenic compounds.

Optimized Systems

Standardized assay formats that assure reproducibility and ease of use

Fluorescence Blocking Membrane

Unique, quantitative platform for analyzing cell migration and invasion

Reproducible and simplified assays

No need for manual cell scraping and counting

Fewer handling steps reduce risk of contamination

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Automation Compatible

Increases throughput and productivity

Fits most robots and fluid handlers

Pre-qualified BD™ HUVEC-2 Cells

Qualified for use in the BD BioCoat Angiogenesis System: Endothelial Cell Migration

Pre-screened for responsiveness to VEGF

Excellent viability

Lot-to-lot consistency

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Advantages of the BD BioCoat™ Angiogenesis Systems

BD BioCoat™ Angiogenesis Systems

A portfolio of products

designed to provide

researchers with

standardized in vitro

assays for quantitative

analysis of key steps in

the angiogenesis process.

bdbiosciences.comUnless otherwise specified, all products are for research use only. Not intended for use in diagnostic or therapeutic procedures. All applications are either tested in-house or reported in the literature. See Technical Data Sheets for details.

�

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00.1 ng/ml 1.0 ng/ml 10 ng/ml

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During angiogenesis, endothelial cells

are activated and express matrix

metalloproteinases (MMPs), which

degrade the vascular basement membrane

and facilitate cellular movement towards

angiogenic stimuli.�-5 The BD BioCoat™

Angiogenesis System: Endothelial Cell

Invasion provides an in vitro, quantitative

assay for prospective anti- and pro-angio-

genic compounds. It is composed of a

BD Falcon™ �4-Multiwell Insert Plate (and

a non-tissue culture (TC)-treated �4-well

receiver plate and lid) containing a

BD FluoroBlok™ fluorescence-blocking,

microporous (�.0 µm pore size) polyethylene

terephthalate (PET) membrane coated with

BD Matrigel™ Matrix. BD Matrigel Matrix is a

reconstituted basement membrane prepara-

tion derived from Engelbreth-Holm-Swarm

(EHS) mouse tumors. 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 endothe-

lial cells with invasive capacity. Following

activation by angiogenic factors, the endo-

thelial cells express MMP � 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.�,4

Unlike traditional in vitro cell invasion assays,

the BD BioCoat Angiogenesis System 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

more than 99 percent of the excitation

and emission wavelengths of fluorophores

commonly used to label cells (Figure 1).

This reproducible system routinely yields

inter- and intra-assay CVs of ≤ 15 percent.

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 demon-

strated using known anti-angiogenic agents

(Figure 2).

Figure 2: HMVECs were assayed in the BD BioCoat™ Angiogenesis System: Endothelial Cell Invasion in the presence of VEGF (4 ng/ml) with varying concentrations of TIMP-2 (=Tissue Inhibitor of Matelloproteinase 2, left) or 1’10’ phenanthroline (right) 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 then analyzed for invasion through BD Matrigel™ Matrix using an Applied Biosystems CytoFluor® 4000 plate reader [485/530 nm (Ex/Em) wavelengths]. Data represents the mean of n=3 inserts ± S.D.

Effects of TIMP-2 and 1’10’ Phenathanthroline in VEGF-Mediated HMVEC InvasionLabeling Cells Post-Invasion with Calcein AM

Figure 1: Fluorescence plate reader quantifies cells post-invasion by measuring fluores-cence and correlating to cell number. Cells on top of the BD FluoroBlok™ membrane, i.e. non-invasive cells, are not detected by the bottom-reading fluorometer.

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485nmDetector

Emission@

530nm

Calcein AM

Analyze endothelial cell

invasion using fluorescence

detection in a simplified

and reproducible manner.

BD BioCoat™ Angiogenesis System:Endothelial Cell Invasion


All applications are either tested in-house or reported in the literature. See Technical Data Sheets for details.4

Cell migration (or chemotaxis) is the

directional movement of cells in

response to a concentration gradient

of a soluble chemoattractant. During angio-

genesis, activated endothelial cells invade

through the basement membrane and

migrate towards a variety of pro-angiogenic

factors.�

The BD BioCoat™ Angiogenesis System:

Endothelial Cell Migration is composed of a

BD Falcon™ �4- or 96-Multiwell Insert Plate

(and a non-TC-treated �4- or 96-well receiver

plate and lid) containing a BD FluoroBlok™

fluorescence-blocking microporous PET

membrane (�.0 µm pore size) evenly coated

with human fibronectin (=HFN) (Figure 3).

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.6 The cells may be labeled with a

fluorescent dye, either pre- or post-migration

(Figure 4). The pre-labeling technique

enables real-time kinetic measurements

of cell migration. Studies conducted using

the post-labeling technique demonstrated

that BD™ HUVEC-� Cells migrate towards

VEGF in a concentration-dependent manner

(Figure 5). 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.7

Figure 4: HUVECs labeled with Calcein AM were placed in the BD BioCoat Angiogenesis System: Endothelial Cell Migration in the presence (bottom) or absence (top) of chemoattractant (VEGF) and incubated over-night. The photographs show a fluorescence signal associated with the underside of the insert membrane.

BD™ HUVEC-2 Cells Exhibit Concentration-Dependent Migration Towards VEGF

HUVEC Cells Reside on the Underside of the Insert Membrane Post-Migration

HUVEC Migration on Uncoated and HFN-Coated Inserts

Figure 3: 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 chemoattrac-tants. 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 results indicate a marked increase in migra-tion 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.

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Fibronectin Coated Inserts

Uncoated Inserts

FBS

A quantitative and repro-

ducible in vitro model for

examining the effects of

prospective compounds on

endothelial cell migration.

Figure 5: BD HUVEC-2 cells assayed in the BD BioCoat Angiogenesis System: Endothelial Cell Migration (96-Multiwell format) in response to increasing concentrations of VEGF. Samples were incubated for 22 hours. Cells were labeled post-migration with Calcein AM and measured by detecting the fluorescence of cells that migrated through the fibronectin-coated BD FluoroBlok membrane with the Victor2™ plate reader (Perkin Elmer) at 485 nm emission. Data represents the mean of n=4 inserts ± S.D.

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BD BioCoat™ Angiogenesis System:Endothelial Cell Migration


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Save time and improve

reproducibility with this

optimized system for

screening compounds that

modulate endothelial cell

tubulogenesis.

Endothelial cells are capable of differen-

tiating in vitro to form capillary-like

structures, or tubes.8,9 Cross-section

analyses of tubes formed in extracellular

matrices such as laminin,10 collagen,9 fibrin,

and BD Matrigel™ Matrix10 revealed the

presence of a lumen surrounded by cells.

When BD Matrigel Matrix is used to promote

endothelial cell tube formation, the lumen-

forming cells interact with each other via

interdigitating junctional complexes.10 This

property is indicative of in vivo-like capillary

formation. Since BD Matrigel Matrix has

been found to induce endothelial cell

tubulogenesis and lumen formation in a time

frame of 18 hours, this matrix is suitable

for high-throughput assays that target this

critical angiogenesis pathway.

The BD BioCoat™ Angiogenesis System:

Endothelial Cell Tube Formation is an in vitro

assay system composed of a BD Falcon™ 96-

well black plate with clear bottom uniformly

coated with BD Matrigel Matrix. To ensure

reproducibility when using this assay system,

different preparations of BD Matrigel Matrix

are screened for the ability to promote

optimal tube formation under standardized

conditions. In addition to the coating

material, the manufacturing process has

been optimized to the highest standards.

Assay performance is further enhanced by

the inclusion of our specially treated 96-

well microplate, which has specific surface

properties that assure even coating and

minimize meniscus formation.

The 96-well format allows for increased

productivity in this traditionally low-

throughput assay. Throughput can be further

augmented through the use of automated

imaging instrumentation to obtain optimal

microscopic images for rapid quantification

of tube formation (e.g. the BD Pathway™

Bioimager). A number of human endothelial

cell types have been shown to form tubes

in the BD BioCoat Angiogenesis System:

Endothelial Cell Tube Formation (Figures 6 and 7). Studies have demonstrated that

endothelial cell tubulogenesis is inhibited by

antagonists to integrin subunits a5b111 and

b11� as well as the antibiotic fumagillin.1�

Using the BD BioCoat Angiogenesis System:

Endothelial Tube Formation System, we

have shown that suramin (Figure 8) and

antibodies directed against b1-integrin

(Figure 9) inhibit endothelial cell tube

formation in vitro.

Human Endothelial Cell Types Exhibit Tube Formation

Figure 6: HUVEC, HMVEC, and the human endo-thelial cell line HMEC-1 exhibit tube formation in the BD BioCoat Angiogenesis System: Endothelial Cell Tube Formation. Here, 20,000 cells of each cell type were added to wells containing pre-solidified BD Matrigel™ Matrix. The assay was incubated for 18 hours. Each bar represents the mean of n=32 wells ± S.D.

HUVEC HMVEC HMEC-1

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Confocal Imaging of BD™ HUVEC-2 Cell Tube Formation

Figure 7: BD™ HUVEC-2 cells (Cat. No. 354151) were assayed using the BD BioCoat Angiogenesis System: Endothelial Cell Tube Formation (Cat. No. 354149). Cells were stained using the fluorogenic esterase substrate Calcein AM (Molecular Probes C1430). Confocal images were captured using the BD Pathway™ Bioimager equipped with a 4x Olympus objective. Please refer to BD Biosciences’ application note

“An Image-Based Assay of Endothelial Cell Tube Formation as a Model of Angiogenesis” for further details.

Suramin Inhibits HMEC-1 Tube Formation

Figure 8: HMEC-1 cells (40,000 cells/ml) were treated with Suramin at concentrations ranging from 0-40 µM and then analyzed for tube formation using the BD BioCoat Angiogenesis System: Endothelial Cell Tube Formation. 50 µl of cells plus compound were added to wells containing pre-solidified BD Matrigel™ Matrix. Samples were incubated at 37°C/5% C02 for 18 hours before staining with Calcein AM. Images were acquired with a 2x objective lens and the total tube length was measured using MetaMorph® (Universal Imaging Corporation™). Each bar represents the mean of n=8 wells ± S.D.

Anti-b1 Integrin Antibody Inhibits HMEC-1 Tube Formation

Figure 9: HMEC-1 cells were incubated in the absence or presence of antibody directed against b1-Integrin and analyzed for tube formation using the BD BioCoat Angiogenesis System: Endothelial Cell Tube Formation. Samples were incubated for 24 hours at 37ºC before staining with Calcein AM. Images were acquired with a 4x objective lens and the total tube length was measured using MetaMorph® (Universal Imaging Corporation™). Each bar represents the mean of n=20 wells ± S.D.

BD BioCoat™ Angiogenesis System: Endothelial Cell Tube Formation

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All applications are either tested in-house or reported in the literature. See Technical Data Sheets for details.6

BD™ Human Umbilical Vein Endothelial Cells

Since commercially available primary

endothelial cells exhibit inherent lot-

to-lot variability, researchers are often

burdened with the need to screen lots for

performance in their cell-based assays.

BD Biosciences has eliminated this difficult

and expensive process by offering pre-

qualified primary endothelial cells.

BD™ Human Umbilical Vein Endothelial Cells

(HUVEC-�) are derived from single donors

and cryopreserved at passage number �

(Figures 10 and 11). BD HUVEC-� Cells

have been pre-qualified to assure a robust

migratory response to angiogenic factors

such as VEGF and FBS. Single donor primary

HUVEC-� cells are suitable for use in

combination with BD BioCoat™ Angiogenesis

Systems to provide relevant models for

angiogenesis (e.g., cardiovascular, vascular,

and wound healing) and cancer research.

In addition to saving time and labor, this

advance screening eliminates the uncertainty

associated with the cell selection process.

BD™ HUVEC-� Cells offer:

• Cell viability ≥ 70%

• 5x105 cells that were cryopreserved at

passage number � in medium containing

10% DMSO in each vial

• Lot-to-lot consistency

• Positive immunohistochemical staining

for von Willebrand factor (vWf) and CD�1

antigen

• Negative immunohistochemical staining

to a-actin

• Positive Dil-Ac-LDL uptake

• Pre-qualification for responsiveness

to VEGF

• Pre-screening for angiogenesis studies

– Qualified for use in BD BioCoat

Angiogenesis System: Endothelial

Cell Migration

– May be used in BD BioCoat

Angiogenesis Systems: Endothelial Cell

Invasion and Tube Formation

Pre-qualified primary

endothelial cells ensure

assay performance and

data reproducibility.

Figure 10: BD™ HUVEC-2 Cells as secondary culture. Positive immunohistochemical staining for CD31 antigen and nuclear DNA using DAPI.

Figure 11: Phase contrast micrographs of BD™ HUVEC-2 Cells at day 3 (left) and day 7 (right) of secondary culture.


7

BD BioCoat™ Angiogenesis Systems for Endothelial Cell Invasion, Migration, and Tube Formation offer standardized formats that increase quality and reproducibility of compound screening assays.

Quality ControlBD BioCoat™ Angiogenesis SystemsAll lots of BD BioCoat Angiogenesis Systems are tested and found negative for bacteria and fungi.

BD™ HUVEC-2 CellsTested for its ability to produce a robust migratory response in response to VEGF. All cells aretested and found negative for HIV-1, mycoplasma, hepatitis B, hepatitis C, yeast, and fungi.

Storage and StabilityAll lots of BD BioCoat Angiogenesis Systems are shipped on dry ice when overnight delivery is possible. Upon receipt, store immediately at -�0°C.BD HUVEC-� Cells are shipped on dry ice. Upon receipt, store immediately in liquid nitrogen.

Ordering Information

Discover the Advantages and Ease of Screening for Pro- and Anti-Angiogenic Compounds

BD BioCoat™ Angiogenesis System: Endothelial Cell Invasion

24-Multiwell Insert System, 3.0 µm, coated with BD Matrigel™ 1 354141 24-Multiwell Insert System, 3.0 µm, coated with BD Matrigel™ 5 354142

description react qty./pack cat.no.

BD BioCoat™ Angiogenesis System: Endothelial Cell Migration

24-Multiwell Insert System, 3.0 µm, coated with Fibronectin 1 354143 24-Multiwell Insert System, 3.0 µm, coated with Fibronectin 5 35414496-Multiwell Insert System, 3.0 µm, coated with Fibronectin 1 354147 96-Multiwell Insert System, 3.0 µm, coated with Fibronectin 5 354148


BD™ HUVEC-2 Cells

Cryopreserved Human Umbilical Vein Endothelial Cells 0.5 Mio. Cells 354151


BD BioCoat™ Angiogenesis System: Endothelial Cell Tube Formation

BD Optilux™ 96-well Black/Clear Microplates, coated with BD Ma 1 354149 BD Optilux™ 96-well Black/Clear Microplates, coated with BD Ma 5 354150

description react qty./pack cat.no.BD Falcon™ FluoroBlok™ 24-Multiwell Insert Systems

One Multiwell Insert Plate in a 24-well Plate, 3.0 µm 1 351155 Five Multiwell Insert Plate in 24-well Plates, 3.0 µm 5 351156


BD Falcon™ FluoroBlok™ 96-Multiwell Insert Systems

One Multiwell Insert Plate in a 96-square well Plate, 3.0 µm 1 351161 Five Multiwell Insert Plate in a 96-square well Plate, 3.0 µm 5 351162


References

1. Auerbach, R. et al., Clinical Chemistry, 49:�� (�00�).

�. Taraboletti, G. and Giavazzi, R., European J. Cancer, 40:881 (�004).

�. Harris, S.R., et al., In Vivo, 12:56� (1998).

4. Benelli, R., et al., Int. J. Biol. Markers, 14(4):�4� (1999).

5. Molema, G., Pharmacological Reviews, 52(2):��7 (�000).

6. Goldberger, A. and Septak, M., BD Biosciences – Discovery Labware, Technical Bulletin #4�8 (1998).

7. Mastyugin, V., et al., J. Biomol. Screening,9:71� (�004).

8. Folkman, J. and Haudenschild, C., Nature, 288:551 (1980).

9. Montesano, R., Orci, L. and Vassalli, J., Cell. Biol., 97:1648 (198�).

10. Kubota, Y., Kleinman, H.K., Martin G.R., and Lawley, T.J., J. Cell Biol., 107:1589 (1988).

11. Kim, S., Bell, K., Mousa, S.A., and Varner, J.A., Am. J. Pathol., 156:1�45 (�000).

1�. Kubota, Y., Kawa, Y., and Mizoguchi, M., J. Dermatol. Sci., 12:�6 (1996).

1�. Ingber, D., et al., Nature, 348:555 (1990).

BD BioCoat™ Angiogenesis System: Endothelial Cell Invasion

Tested for its ability to allow invasion of Human Microvascular Endothelial Cells (HMVEC-1), a human microvascular endothelial cell line, and to exclude invasion of �T� cells, a non-invasive fibroblast cell line.

BD BioCoat™ Angiogenesis System: Endothelial Cell Migration

Tested for its ability to support migration of Human Umbilical Vein Endothelial Cells (HUVEC) in response to Vascular Endothelial Cell Growth Factor (VEGF).

BD BioCoat™ Angiogenesis System: Endothelial Tube Formation

Tested for its ability to support HMVEC tubule formation, determined by tubule length, and measured by automated image analysis.

BD BioCoat™ & BD Falcon™ FluoroBlok™ Individual Inserts

24-well size Individual Inserts, 3.0 µm, coated with Fibronectin 24 354597 24-well size Individual Inserts, 3.0 µm, uncoated 48 351151


BD BioCoat™ Endothelial Cell Growth Environment

Endothelial Cell Growth Environment (75 cm2 Flasks) 1 kit 355053 Endothelial Cell Culture Medum, incl. Additives 500 ml 355054Endothelial Cell Growth Supplement, Biovine 15 mg 354006Endothelial Cell Growth Supplement, Biovine 100 mg 35600675 cm2 Plug-Seal Flasks, coated with Collagen I 5 35446275 cm2 Vented-Cap Flasks, coated with Collagen I 5 354485


BD™ Cytokines

VEGF, Human Recombinant, E.coli 10 µg 354107 bFGF, Human Recombinant, E.coli 10 µg 354060bFGF, Bovine Natural 10 µg 356037


For Research Use Only. Not intended for use in diagnostic or therapeutic procedures. CytoFluor is a registered trademark of Applied Biosystems.MetaMorph is a registered trademark of Universal Imaging Corporation™ (UIC).BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2006 BDXEUR4159-00

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BD BioCoat Angiogenesis Systems