| Assay Method Information | |
| | Arrayscan Assay |
| Description: | U2OS cells (1500 per well) were plated in 40 μL of DMEM media (containing 10% fetal calf serum and 2 mM Glutamax T-1) in costar 384-well plates and left overnight at 37° C. and 5% CO2 to adhere.Cells were then dosed with compound diluted in DMSO (120 nL added to each well to give 0.0313-30 μM concentrations of compound) and incubated at 37° C. and 5% CO2. After 1 h of treatment with compound, all wells except min wells were dosed with 17-AAG diluted in DMSO (10 nL added to each well to give 250 nM final concentration) and plates were incubated overnight at 37° C. and 5% CO2. The following day the cells were fixed by addition of 20 μL/well of 12% formaldehyde with 1:1700 Hoechst in PBS for 10 min at room temperature. The fixative was decanted and the wells washed once with 50 μL of phosphate buffer saline (PBS). The PBS was subsequently aspirated, and the cells were permeabilized by addition of 20 μL/well of PBS 0.3% Triton X-100 for 20 min at room temperature. The wells were then washed with 80 μL of PBS prior to the addition of 20 μL of combined primary and secondary antibodies diluted in PBS (1:10 000 mouse anti-Hsp72 #SPA-810 purchased from Stressgen and 1:3000 Alexa Fluor 488 goat anti-mouse IgG (H+L) #A-11001 molecular probes), for 2 h at room temperature. The wells were then washed with 50 μL of PBS. Finally, 50 μL of PBS was added to each well, and the plates were sealed ready to analyze. Analysis was carried out using a Cellomics Arrayscan VTI instrument and the Cellomics Arrayscan Compartmental Analysis algorithm to measure cellular levels of HSP72. Results are reported as IC50 values for inhibition of HSP72 levels |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |