Victor 3v 1420 multilabel plate reader

To monitor the sensitivity of RET cells or TT cells to chemotherapy cell viability assays were performed. Empty vector (EV, control) cells along with cells containing RET 9 and RET 51 isoforms were plated in 96-well plates (10K/well) and allowed to adhere overnight. Medium was replaced with medium containing doxorubicin (DOX) (D1515, Sigma Aldrich, St. Louis, MO) at 0 nM, 100 nm, 250 nM and 500nM. TT cells were also plated in 96-well plates (10K/well) and allowed to adhere overnight before DOX was added to fresh medium at 100nM. In assays looking at the effect of DNAPKcs inhibition, cells were treated with NU7441 inhibitor (KU-57788, Selleckchem, Houston, TX) with or without DOX at 1.0 and 2.0 μM (1 μM for TT cells). After 24 hours, plates with RET cells were removed from the incubator and measured for cell viability using the CellTiter-Glo Luminescent Cell Viability Assay kit (# G7572 Promega, Madison, WI) according to the manufacturer’s protocol. Due to the slow growth rate of TT cells, the assays were performed 4 days after drug was applied. Luminescence was measured on a Perkin Elmer Victor 3V 1420 Multilabel plate reader. The assay was performed in triplicate and statistical analysis performed by standard ttests.

Medium was removed and cells were washed twice with PBS. The cells were lysed in ALP lysis buffer (10 mM glycine; 0.1 mM MgCl2; 0.01 mM ZnCl2; pH 10.5; 0.1% Triton X-100) for 3 hours. The assay was performed with 25 μl per sample with 2 μl of ALP substrate (6 mM p-nitrophenylphosphate, PNPP) and 198 μl of ALP assay buffer (100 mM glycine; 1.0 mM MgCl2; 0.1 mM ZnCl2; pH 10.5). ALP activity was directly measured at 405 nm with a Victor3V, 1420 Multilabel plate reader (Perkin Elmer, NL) for 120 minutes, with measurements every 5 minutes. The slopes of the curves were compared between controls and samples treated with 5 mM D-2-HG.