Human Endothelial Cell Proliferation Assay - Dose Response
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 (Carmeliet, 2003). Targeting angiogenesis has recently emerged as a proven therapeutic strategy for treatment of cancer and age-related macular more ..
BioActive Compounds: 576
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 (Carmeliet, 2003). Targeting angiogenesis has recently emerged as a proven therapeutic strategy for treatment of cancer and age-related macular degeneration (Coultas, et al, 2005, Carmeliet, 2005). However, more research is needed to understand biological signaling pathways of angiogenesis and mechanisms of endothelial activation for target identification 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. We had previously reported the results of a high throughput screening of the MLSCN compound library against two primary human cell lines, endothelia (HUVEC) and fibroblasts (LL 47) (AID 648 & 719, respectively). Primary hit compounds with differential inhibitory activity against endothelial cell activation had been selected based on the screening results. To assess potency of the identified primary hit compounds against human endothelial cell activation, a study of dose response with ten doses ranging from 0.039 uM to 20 uM in HTS setting has been completed and EC50 values on inhibition HUVEC growth have been determined based on the dose response curve of each hit compound.
Cells- Human umbilical vein endothelial cells (HUVEC)-Clonetics.
Growth medium- Endothelial Cell Basal Medium (EBM) supplemented with 2% fetal bovine serum, 12 ug/mL bovine brain extract, 1 ug/mL hydrocortisone, and 1 ug/mL GA-1000 (gentamicin-amphothericin).
Conditions- 37C with 5% CO2 and 95% humidity. Cells are maintained for no more than 12 passages.
Plates-Corning Flat Clear Bottom Black Polystyrene TC-Treated (Cat. No.:3712)
Collagen (Sigma Cat. no.: C9791)
Preparation of collagen solution
Dissolve collagen in 1 mM acetic acid (0.5 mg/mL).
Filter the solution through a 0.22 um PES filter and store at 4C.
Collagen treatment of plates
Dispense 25 uL collagen solution into 384-well plates.
Incubate at least 1 h at room temp.
Remove collagen solution from plates with Biomek FX.
Centrifuge plates upside down to spin out any residual liquid (2000 RPM for 5 min).
Dry plates in laminar flow hood (sterile conditions).
Store at 4C for up to two weeks.
Plate 1250 cells per well in 20 uL media (62,500 cells/ml) with Matrix WellMate.
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 h.
Transferred entire bin to the incubator and crack the lid open.
Incubate at 37C with 5% CO2 and high humidity. The lid on the pan must remain vented while incubating.
Dilution media, carrier and positive control drug were dispensed to dilution plate. Compounds were diluted in complete growth medium to prepare a 10x concentrated dosing solution (0.39-200 uM). A volume of 2.5 uL of the diluted compounds was transferred to the cell plate using a Biomek FX and the plates were returned to the incubator. This resulted in final culture volume in assay plates of 25.5 uL, with final concentrations of 1 ug/mL 6-methyl purine riboside (MePR) and 0.039 to 20 uM for library compounds. DMSO concentrations were maintained at 0.2% in all wells.
Cell viability measurement:
After incubating 72 h at 37C, assay plates were removed from the incubator and equilibrated to room temperature. As per the manufacturer's protocol, an equal volume of CellTiter-Glo reagent (Promega Inc.) was added to each well and plates were incubated for an additional 10 min at room temperature. At the end of the incubation, luminescence was measured using an EnVision multi-label reader (Perkin Elmer) with an integration time of 0.1 s.
Thirty-two control wells containing cells only and 32 wells containing MePR were included on each assay plate and used to calculate Z value for each plate and to normalize the data on a per plate basis. Z values ranged from 0.7 to 0.82 with a mean of 0.76. Compound data was normalized and reported as % viability which was calculated using the following formula: % viability = 100 x luminescence compound well / median luminescence cell control. The normalized % viability was plotted against the tested concentrations of 0.039 to 20 uM . The EC50 values were calculated using XLfit formula 205, a 4 parameter Levenburg-Marquardt algorithm allowing extrapolation up to 100 uM.
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.
Outcome: Compounds with EC50 values <= 100 uM were considered Active within the context of this assay. Compounds with HUVEC specific activity were determined by comparing the activity score derived in this assay with the activity score derived in the LL 47 based assay (AID 821).
Score: In this confirmatory dose response screen active compounds were scored on a scale of 41-80 using an inverse linear correlation to EC50s between 0 and 100 uM. Compounds that did not confirm as actives in the dose response screen were given the score 0. In later stage probe development screening, active resynthesized confirmatory screen compounds and active analogues thereof will score in a range of 81-100.
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Data Table (Concise)