In situ hdac activity fluorometric assay kit

Total HDAC activity was assessed with the in situ HDAC activity fluorometric assay kit (Sigma Aldrich, St. Louis, MO). After isolation, kidney endothelial cells were incubated in plasma from the same donor rat for one hour in a 37°C water bath. Cells were then centrifuged 1000×g for 5 minutes at room temperature and resuspended in the reaction mix (89% HBSS, 10% plasma, 1% substrate). Cells were plated at 50,000 cells/well in a 96 well plate. The reaction proceeded for 2 hours at 37°C in presence of either 300 nM MS-275 (Cayman Chemical, Ann Arbor, MI) or vehicle (0.01% DMSO). After 2 hours, the deacetylated substrate was developed following manufacturer instructions. Each condition was assayed in duplicate. The portion of activity inhibited by MS-275 was attributed to HDAC1. MS-275 is a class I HDAC inhibitor with an IC₅₀ of 300 nM and 8 μM for HDAC1 and HDAC8 respectively.^{23 (link)} Signal from background wells containing only the reaction mix was subtracted from each sample. The amount of generated fluorescent product was calculated using a standard curve generated with the provided standards and divided by 120 minutes to calculate the activity in pmol/min. Vehicle treated samples were considered total HDAC activity. The activity of MS-275 treated samples was subtracted from those treated with vehicle to calculate HDAC1 activity.