Human Endothelial Cell Proliferation Assay
Angiogenesis is a process of new blood vessel formation. Endothelial cell proliferation is an essential step during the angiogenesis process and is involved in many human diseases including cancer, diabetic retinopathy, and rheumatoid arthritis. Targeting endothelial cell proliferation is an attractive strategy for the development of novel therapeutics; although more research is needed to better understand the mechanisms of endothelial cell growth regulation and to identify targets for the discovery of selective angiogenesis modulators. ..more
BioActive Compounds: 126
Southern Research Molecular Libraries Screening Center (SRMLSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Screening Centers Network (MLSCN)
Assay Provider: Dr. Zhican Qu, Southern Research Institute
Angiogenesis is a process of new blood vessel formation. Endothelial cell proliferation is an essential step during the angiogenesis process and is involved in many human diseases including cancer, diabetic retinopathy, and rheumatoid arthritis. Targeting endothelial cell proliferation is an attractive strategy for the development of novel therapeutics; although more research is needed to better understand the mechanisms of endothelial cell growth regulation and to identify targets for the discovery of selective angiogenesis modulators.
The goal of this high throughput screening campaign is to identify compounds that inhibit endothelial cell proliferation to serve as chemical probes for angiogenesis research. Here we describe a cell-based assay using primary cultures of human vascular endothelial cells to screen the NIH Small Molecule Repository. Future plans will be to evaluate the activity of hits on a human fibroblast line to differentiate non-selective inhibitors from selective inhibitors of endothelial cell proliferation, which are anticipated to provide useful research tools and potential leads for antiangiogenic drug discovery.
Cells- Human umbilical vein endothelial cell (HUVEC)-Clonetics (San Diego, CA).
Growth media- Endothelial Cell Basal Medium (EBM) supplemented with 2% fetal bovine serum (FBS), 12 ug/ml bovine brain extract, 1 ug/ml hydrocortisone, and 1 ug/ml GA-1000 (gentamicin-amphothericin).
Conditions- 37C with 5% C02 and 95% humidity. Cells are maintained for no more than 12 passages.
Plates-Corning BCBTCT (Part Number:3712)
Collagen (Sigma Cat: C9791)
Preparation of collagen solution
Dissolve collagen (Sigma Cat: C9791) in 1mM acetic acid (0.5mg/ml).
Filter the solution through a 0.22 micron PVDF filter and store at 4C.
Collagen treatment of plates
Dispense 25uL Collagen solution into 384 well plates.
Incubate at least 1 hr at room temp.
Remove collagen solution from plates with Biomek FX.
Centrifuge plates upside down to spin out any residual liquid (2000RPM for 5 minutes).
Dry plates in laminar flow hood (sterile conditions).
Store at 4C for up to two weeks.
Plate 1250 cells per well in 20ul media (62,500/cells per ml) with Matrix WellMate and a wide bore cassette head.
Removed from the dispenser in groups of five.
Tap stack on all four sides to flatten the meniscus and evenly distribute the cells.
Transferred to benchtop bins.
Incubate at RT for 1 hour.
Transferred entire bin to the incubator and crack the lid.
Incubate at 37C with 5% CO2 and high humidity. The lid on the pan must remain vented while incubating.
Dilute compounds to 50uM (5x) in growth media with Biomek FX.
Add 5ul to assay plate for final conc. of 10uM (0.1% DMSO) with FX.
Cell control-growth media with 0.1% DMSO.
Drug control-6-methyl purine riboside (MePR) 1ug/ml
Drug plates in groups of 4-5 plates to minimize time spent out of the incubator
Incubate at 37C with 5% CO2 and high humidity for 72 hours
Equilibrate to room temp for 30 min.
Read CTG endpoint as per manufacturers protocol.
32 cell control wells and 32 drug control wells are included on each assay plate and are used to calculate Z values for plate QC and to normalize the data on a per plate basis.
Results are reported as % viability and are calculated using the following formula:
% viability = luminescence compound well / median luminescence cell control*100
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.
The compounds that reduced cell viability in both assays 30% were assigned an outcome of Active. Compounds that reduced cell viability in only one assay to less than or equal to 30% were scored as Inconclusive. Compounds with %viability between 100 and 30% were scored as Inactive.
Because of the inherent error in all high throughput screens, compounds that were active in this single dose screen were assigned a score of 100. All other compounds were assigned a score of 0.
Data Table (Concise)