Affordable Luciferase Reporter Assay for Cell-Based High-Throughput Screening

Abstract

The firefly luciferase gene is commonly used in cell-based reporter assays. Convenient luciferase assay reagents for use in high-throughput screening (HTS) are commercially available. However, the high cost of these reagents is not within the means of some academic laboratories. Therefore, we set out to develop an affordable luciferase assay reagent applicable in an HTS format using simple liquid-handling steps. The reagent was homemade from individual chemical components and optimized for luminescence intensity and stability. We determined the minimal concentrations of the most expensive components, dithiothreitol (DTT) and D-luciferin, resulting in a total assay reagent cost of less than 1 cent per sample. Signal stability was maximized by omission of coenzyme A and reduction of DTT concentration. The assay was validated in a high-throughput setting using two cancer cell lines carrying a p53-dependent luciferase reporter construct and siRNAs modulating p53 transcriptional activity. Induction of p53 activity by silencing PPM1D or SYVN1 and reduction of p53 activity by silencing p53 remained constant over a 2-h measurement period, with good assay quality (Z′ factors mostly above 0.5). Hence, the luciferase assay described herein can be used for affordable reporter readout in cell-based HTS.

Biosensor‐assisted engineering of a high‐yield Pichia pastoris cell‐free protein synthesis platform

“…A luciferase assay mix (20 mM tricine, 100 μM EDTA, 1.07 mM MgCO 3 , 250 μM luciferin, 250 μM ATP, 2.67 mM MgSO 4 , and 17 mM DTT; Siebring‐van Olst et al, ) was made fresh before the assay. Every 30 min, 5 μl of the cell‐free reaction was added to 30 μl of the luciferase assay mix and the luminescence was measured using the POLARstar ® Omega plate reader (BMG Labtech Ltd, Aylesbury, UK).…”

The Cytidine Analog Fluorocyclopentenylcytosine (RX-3117) Is Activated by Uridine-Cytidine Kinase 2

“…The cells were given 24 hr to reattach to the plates after which mixes of siRNAs with dharmaFECT 1 transfection reagent (GE Dharmacon) were added to the plates as described earlier [14], 20 μl for 96-well and 600 μl for 6-well plates. The mixes required for transfection of 1,000 cells in 80 μl of medium consisted of 19.45 μl serum free medium, 0.5 μl of 5 μM siRNA and 0.05 μl of transfection reagent and were prepared as specified by the manufacturer.…”

Whole genome RNAi screens reveal a critical role of REV3 in coping with replication stress

“…High‐throughput screening (HTS) was done essentially as described previously (Siebring‐van Olst et al., 2013). One day before siRNA transfection, S1C6 or R1B6 cells (750 cells per well) were seeded in 272 transparent flat‐bottom 96‐well plates (TPP, Switzerland) in 80 μl of complete culture medium.…”