| Assay Method Information | |
| | MLKL Phosphorylation High-Content Assay |
| Description: | HT29-L23 human colorectal adenocarcinoma cells were maintained in RPMI 1640 medium containing 10% heat-inactivated FBS, 1% Penicillin-Streptomycin and 10 mM HEPES. Cells were seeded at 2,000 cells/well in 384 well tissue culture -treated microplates (Greiner # 781090-3B) and incubated at 37 °C (5% CO2/95% O2) for 2 d. On the day of the assay, the cells were treated with test compounds at final concentrations of 6.25 to 0.106 μM for 30 min at 37 °C (5% CO2/95% O2). Necroptopsis was induced using a mixture of human TNFα (35 ng/mL) (Peprotech #300-01 A), SMAC mimetic (from US 2015/0322111 Al) (700 nM) and Z-VAD (140 nM) (BD pharmingen #51-6936). Following 6 h incubation at 37 °C (5% CO2/95% O2), the cells were fixed with 4% formaldehyde (ACROS 11969-0010) for 15 min at rt, then permeabilized with phosphate buffered saline (PBS) containing 0.2% Triton-X-100 for 10 min. MLKL phosphorylation was detected using anti-MLKL (phospho S358) antibody (Abeam #abl87091) (1 : 1000 dilution in Blocking Buffer [PBS supplemented with 0.1% BSA]) with ON incubation at 4 °C. After washing three times in PBS, goat anti-rabbit Alexa- 488 (1 : 1000 dilution) (Life Technologies, Al 1008) and Hoechst 33342 (Life Technologies, H3570) (1 :2000 dilution) in Blocking Buffer were added for 1 h at rt. Following another three cycles of washes in PBS, the microplates were sealed, and cellular images were acquired in the Cellomics ArrayScan VTI high-content imager equipped with an XI camera. Fluorescent images were taken using a lOx objective and the 386-23 BGRFRN BGRFRN and 485-20 BGRFRN BGRFRN filter sets, for nuclei and MLKL phosphorylation, respectively. The image sets were analyzed using the Compartmental Analysis Bioapplication software (Cellomics). The level of MLKL phosphorylation was quantified as MEAN CircRingAvglntenRatio. The maximal inhibitory response was defined by the activity induced by Neels (CAS #: 852391-15-2, 6.25 pM). The IC50 value was defined as the concentration of compound that produces 50% of the maximal inhibition. The data were fitted using the 4-parameter logistic equation to calculate the IC50 and Ymax values. |
| Affinity data for this assay | |
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