Fluorogenic hdac assay kit

Enzyme-active recombinant HDAC4, 7 and 8 were purchased from BPS Bioscience Inc., and HDAC2 was purchased from BIOMOL. The HDAC4 and HDAC7 assays were performed by Fluorogenic HDAC Class 2a Assay Kit (BPS Bioscience), and the HDAC2 and HDAC8 assays were performed by Fluorogenic HDAC Assay Kit (BPS Bioscience). The specific HDAC fluorometric substrate, comprised of an acetylated lysine side chain, was incubated with purified individual HDAC enzymes (HDAC2, 4, 7 and 8), in the presence or absence of the HDAC inhibitors, PBA, BS, TSA or VAL, at different doses. After adding Lysine Developer, the fluorophore that could then be measured using the fluorescence plate reader Spectra (Max Gemini, Molecular Devices, Sunnyvale, CA), was excitated at a wave-length of 365 nm and detected by emitted light at 450 nm.

A fluorogenic HDAC assay kit (BPS Bioscience, San Diego, CA, USA) was used to assess the ability of butyrate to inhibit enzyme activity of recombinant class I HDAC according to the manufacturer’s instructions. Briefly, HDAC was incubated with butyrate (100, 500, 1000, 2000 µM) on 96-well plates for 1 h. HDAC substrate was added (5 μL of 1 mM DMSO stock solutions), and the plates were incubated at 37 °C for 30 min. Finally, developer solution was added for an additional 15 min at 37 °C.
To measure inhibition of HDAC activity by butyrate in NPICCs, an in situ HDAC activity fluorometric assay (BioVison, Milpitas, CA, USA) was used. One hundred NPICCs were seeded in 96-well plates and cultured in BI-C medium or medium supplemented with 1000 µM butyrate. After six days, the medium was removed and reaction mix (100 µL per well) was added to each well for 3 h at 37 °C. Then, 100 µL developer solution was added into each well for 30 min at 37 °C. Fluorescence signals were detected using a FLUOstar^® Omega Plate Reader (BMG LABTECH, Ortenberg, Germany), with excitation and emission filters of 355 nm and 460 nm. All the measures were performed in duplicates or triplicates.