Prl tk renilla luciferase control vector

HEK293T cells were passaged as previously described and plated in a 24 well Nunclon delta surface plate at a density of 2×10⁵ cells in 500μl media (as described above). Cells were incubated at 37°C/5% CO₂ for 2 hours prior to transfection. Transfection reactions (one per well) were prepared as follows: 500 ng of pGL3 reporter plasmid (or an equimolar equivalent of a MERTK sequence containing plasmid) was added to 100μl of OPTI-MEM (1×) reduced serum medium. To control for variable levels of transfection between reactions, cells were co-transfected with 5 ng (1 : 100) of a pRL-TK Renilla luciferase control vector (Promega) and mixtures were incubated for 15 minutes at room temperature. Following incubation, 2μl of Lipofectamine LTX reagent (Invitrogen, CA) was added and mixtures were incubated at room temperature for a further 25 minutes. Mixtures were added to each well and cell lysates were harvested after 48 hours using the Promega Dual-Luciferase reporter 1000 assay system (Promega, USA) in accordance with manufacturer’s instructions. Quantification of firefly and renilla luciferase activity was measured with a POLARstar OMEGA microplate reader equipped with two injectors (BMG Labtech, Germany) in accordance with manufacturer’s instructions.