Cell-Based HDAC Assays
Cell-based HDAC experiments are especially relevant because they address the natural context of HDAC enzymes and can reflect the diversity of expression arising from different cell types. Furthermore, on-target HDAC potency can be linked to functional outcomes such as on- and off-target toxicities. Because the HDAC-Glo I/II Substrate is cell permeable, the assay can be conducted in either a lytic (with the addition of detergent) or nonlytic manner. Nonlytic formats are particularly attractive when the cell source is semi-precious and too difficult to obtain for parallel experimentation.
As illustrated in Figure 3, human embryonic stem cells (hESC) were contacted with various HDAC inhibitors for a period of one hour. The HDAC-Glo I/II Reagent was then applied in a nonlytic formulation and HDAC activity measured. Medium and reagent could then be removed for additional experimentation such as RT-PCR.
The hESC contained robust HDAC activity that can be inhibited in a dose-dependent manner and compounds rank ordered. The SIRT-Glo Substrate, while also cell permeable, is not recommended for cell-based applications due to HDAC cross-reactivity with the SIRT-Glo peptide sequence.