Quantifying Nuclear HDAC Activity in RAW246.7 Cells

Nuclear protein of RAW246.7 cells was extracted with nuclear and cytoplasmic extraction kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions, and HDAC activities upon LPS treatment were evaluated by Histone Deacetylase Assay Kit, Fluorometric (Abnova, USA). Briefly, a total of 10–50 μg of nuclear extract was added to each well, then diluted with ddH₂O until the final volume was 85 μL (for background reading, add 85 μL ddH₂O only), or added 2 μL of Trichostatin A into diluted sample for negative control. Besides, 2 μL of HeLa nuclear extract was diluted with 83 μL ddH₂O for positive control. After incubating with HDAC Fluorometric substrate at 37 °C for 30 min, 10 μL of Lysine Developer was added to stop the reaction. The plate was incubated at room temperature for 30 min, the fluorescence intensity of the wells was measured on a fluorometric plate reader with excitation set at 350–380 nm and emission detection set at 440–460 nm.