Softmax microplate reader

Caspase-1 activity was determined by a Caspase-1 Fluorometric Assay (BioVision, Mountain View, CA, USA), according to the manufacturer’s instruction. Briefly, 10⁷ B-lymphocytes were stimulated with Curdlan and CpG in the presence of the caspase inhibitor YVAD-CMK, incubated at 37°C for 24 h and lysed with cell lysis buffer on ice for 10 min. Then, equal volumes of 2× reaction buffer and YVAD-AFC were added to the 200 µg of lysates in a 96-well plate and incubated for 2 h at 37°C. The samples were read on a Softmax microplate reader (Molecular Devices, Sunnyvale, CA, USA) with a 400 nm excitation filter and 505 nm emission filter. The caspase-1 activity is expressed relative to the unstimulated control.

Inhibition of ADP-induced aggregation was assessed in a Costar 96-well flat-bottom plate (Sigma-Aldrich, St. Louis, MO, USA) at room temperature. A 188-µL volume of the diluted platelet was added to each well of the plate along with 2 µL of test compound dissolved in dimethyl sulfoxide (DMSO). The reagents were mixed by placing the plate on a shaker at 1050 rpm for 1 min. A 10-µL volume of 400 μM ADP (final concentration: 20 μM) was added to the wells, and after additional shaking at room temperature for 3 min, the absorption of the samples was read at 595 nm on a Softmax microplate reader (Molecular Devices, Sunnyvale, CA, USA). A dose-response curve was generated using serial dilutions of ADP by adding the vehicle DMSO instead of antagonists in order to calculate the half-maximal effective concentration (EC50) of ADP. Inhibition of aggregation was determined as the increase in absorption at 595 nm after 3 min of incubation relative to absorption values of control preparations (0 and 20 μM ADP without antagonists). Sigmoidal dose-response curves and half-maximal inhibitory concentration (IC₅₀) were derived by non-linear regression analysis using Prism 5.01 software (GraphPad, San Diego, CA, USA).