Cell Death Assay

Cell death was measured by PI incorporation as previously described (33, 34). Briefly, the cells were cultured in 24-well plates overnight and replaced the complete medium for the Opti-MEM with LPS (0.5 μg/ml). Four hours later, the cells were treated with TAS for 1 h followed by stimulation with nigericin (2 μM for TGPMs, 3 μM for BMDMs) for 1 h. Then the PI (2 μg/ml) and Hoechst 33342 (5 μg/ml) mixture were added into the cells medium to stain the dead cells and the nuclei, respectively. The stained cells were observed by live imaging using Zeiss Axio Observer D1 microscope equipped with a Zeiss LD Plan-Neofluar 20×/0.4 Korr M27 objective lens (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). Fluorescence images were captured with a Zeiss AxioCam MR R3 cooled CCD camera controlled with ZEN software (Carl Zeiss).