Hdac activity assay kit

BEAS-2B cells were seeded at a density of 2 × 10⁵/ml. The cells were infected with M. pneumoniae, the supernatant discarded, and intracellular HDAC activity determined using an HDAC activity assay kit (Cayman) according to the manufacturer’s instructions. HDAC activity was calculated using the following formula: HDAC activity (nmol/min/ml) = [corrected sample fluorescence - (y-intercept)/slope] × dilution/15 min.

HDAC activity of class I HDACs (HDAC1, 2, 3 and 8) was measured with the fluorometric HDAC Activity Assay kit: HDAC1 (10011563, Cayman Chemical, Ann Arbor, MI), HDAC2, HDAC 3, and HDAC 8 (50062, 50073 and 50068, BPS Bioscience, San Diego, CA), according to the manufacturer’s instructions. HDAC activity of class II HDACs (HDAC4 and HDAC6) were measured with the fluorometric HDAC Activity Assay kit: HDAC4, HDAC 4 (50064 and 50076, BPS Bioscience, San Diego, CA), according to the manufacturer’s instructions.
Due to interference, concentrations of >30 mM were not used in the IC50 calculation. To account for any interference at lower concentrations, all baseline measurements of blank wells (no HDAC enzyme) were performed and subtracted with the various concentrations of diacetyl.