Human Lung Fibroblast Proliferation Assay
Angiogenesis is the process of new blood vessel formation, which is believed to be involved in many human diseases including cancer, diabetic retinopathy, and rheumatoid arthritis. Endothelial cell proliferation is known to occur in early stages of angiogenesis. As such, the identification of compounds that selectively inhibit endothelial cell proliferation represents an attractive strategy for more ..
BioActive Compounds: 937
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 the process of new blood vessel formation, which is believed to be involved in many human diseases including cancer, diabetic retinopathy, and rheumatoid arthritis. Endothelial cell proliferation is known to occur in early stages of angiogenesis. As such, the identification of compounds that selectively inhibit endothelial cell proliferation represents an attractive strategy for the development of novel drugs, which act by inhibiting angiogenesis. We have previously reported the results of a high throughput screening campaign to identify compounds from the NIH Molecular Libraries Small Molecule Repository (MLSMR) that inhibit the proliferation of human vascular endothelial cells (HUVEC)
This assay was designed to interrogate the MLSMR for compounds that inhibit fibroblast proliferation. Compounds that are inactive in this assay, but active in the HUVEC assay will be evaluated further as potential probes for angiogenesis research. Conversely, compounds that selectively inhibit the proliferation of fibroblasts may have utility for other areas of research such as the study of mechanisms involved in tumor desmoplasia or diseases involving fibrosis.
Cell growth conditions:
Human lung fibroblasts (LL 47:ATCC, cat. no. CCL-135) were grown under standard cell culture conditions involving incubation at 37C with 5% C02 and 95% humidity. Cell were grown in IMEM media (Mediatech Inc, cat. no. 10#24-CV) supplemented with 15% fetal bovine serum (Hyclone, cat. no. SH30071.03) and 50 ug/mL gentamicin (Mediatech Inc, cat. no. 30-005-CR) and referred to as Complete Growth Media. Cells were harvested 1:5 in 100 mm Petri plates twice a week, after washing with PBS, followed by 1 mL of trypsin (Invitrogen, cat. no. 4671). Cells used for this assay were maintained in culture for no more than 12 passages.
Fibroblasts were dispensed using a Matrix WellMate bulk dispenser into 384 well plates (Corning BCBTCT, cat. no. 3712) at a density of 1,150 cells per well (23 uL). Cell suspensions were maintained at room temperature with stirring during the plating procedure. Following plating, cells were incubated at 37C overnight prior to the addition of library compounds.
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 (100 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 10 uM for library compounds. DMSO concentrations were maintained at 0.1% 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 a Perkin Elmer Envision multi-label reader 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 ant to normalize the data on a per plate basis. Results are reported as % viability and were calculated using the following formula: % viability = luminescence compound well / median luminescence cell control x 100.
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.
Compounds that exhibited viability of 50% or less were defined as Active, while compounds that exhibited greater than 50% viability were defined 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)