- Use frozen cells as ordinary, cost-effective assay reagents for screening
- Decouple the labor- and time-intensive production of live cells from your screening process
- Obtain pharmacological profiles similar to those observed with growing cells
- Avoid the variability associated with cell division; get more consistent results
Invitrogen’s Division Arrest technology
(1-4) for G-protein–coupled receptors (GPCRs) provides frozen cells by the plate for convenient, cost-effective screening (Figure 1). Because GeneBLAzer® Division Arrested (DA) cells exhibit response profiles similar to those obtained with GeneBLAzer® dividing cells (Figure 2), you can be confident in the pharmacological data you gather. GeneBLAzer® DA cells can be assayed within 24 hours of thawing, and assay consistency can be maintained over periods of up to five days. Cell numbers for division arrested cells increase only marginally after plating, which removes the variability caused by cell division during the course of an assay and provides more consistent results (Figure 3).
- Cost-Effective Access to High Quality, Validated GPCR Cell Lines
Figure 2 - Dose response to VIP of VPAC1 CHO-K1 non-division-arrested and division-arrested cells. VPAC1 CHO-K1 cells (10,000 cells/well) were plated the day before the assay in a 384-well format in division-arrested and non-division-arrested formats. The day of the assay, cells were stimulated with VIP over the indicated concentration range in the presence of 0.5% DMSO for 3 hr. Cells were then loades with LiveBLAzer™-FRET B/G Substrate (2 μM final concentration of CCF4-AM + 1 mM Solution D) for 2 hr. Fluorescence emission values at 460 nm and 530 were obtained using a standard fluorescence plate reader, and the response ratios plotted against the indicated concentrations of VIP (n=6 for each data point).
Figure 3 - Improved assay consistency with division-arrested cells. Three sets of division-arrested (DA) and growing (GR) cells were plated at the same time but entered the assay at three different time points. Better CV and Z' -factor valutes were observed with division-arrested cells (CV 7.5%, Z'-factor 0.76) than with growing cells (CV 20.1%, Z'-factor 0.38).
GeneBLAzer® GPCR DA cells are stable cell lines derived from our fully validated GeneBLAzer® dividing cells that have been treated with a low dose of mitomycin C. This proprietary treatment causes no apparent toxicity or change in cellular signal transduction. GPCR DA cells are functionally validated using the GeneBLAzer® beta-lactamase readout to determine Z’-factor and EC50 concentrations using a primary agonist (data available upon request). Many cell lines have also been tested using the calcium indicator, fluo-4 NW.
View our complete list of GeneBLAzer® GPCR Division arrested and dividing cell based assays.
Invitrogen acquired Division Arrest technology through our acquisition of Sentigen Holding Corp. As part of the Sentigen transaction, Invitrogen acquired the patent family which includes U.S. Patent No. 7,045,281 and Australian Patent No. 2003270073. Patent applications are currently pending in this family in the United States and a number of other countries.
Screening and Profiling
- SelectScreen™ Functional GPCR Profiling Service
- Fursov, N. et al. (2005) Improving consistency of cell-based assays by using division- arrested cells. Assay Drug Dev Technol 3(1): 7–15.
- Kunapuli, P. et al. (2005) Application of division arrest technology to cell-based HTS: comparison with frozen and fresh cells. Assay Drug Dev Technol 3(1): 17–26.
- Digan, M.E. et al. (2005) Evaluation of division-arrested cells for cell-based high-throughput screening and profiling. J Biomol Screen 10(6): 615–623.
- Vasudevan, C. et al. (2005) Improving high-content-screening assay performance by using division-arrested cells. Assay & Drug Development Technologies 3(5): 515–523.