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ASC–GFP transfection in 293T cells and ASC-GFP speck formation assay

MH Menghang Huang
XZ Xiaoxiao Zhang
GT Gee Ann Toh
QG Qin Gong
JW Jia Wang
ZH Zhifu Han
BW Bin Wu
FZ Franklin Zhong
JC Jijie Chai
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293T ASC–GFP and 293T ASC–GFP DPP8/DPP9 double-knockout cells have been previously described9. All transfections were carried out using Lipofectamine 2000 (Thermo Fisher). For immunoprecipitation, cells were collected 48 h post transfection. For the ASC–GFP speck assay, cells were fixed 24 h post transfection and counterstained with DAPI or Hoescht before wide-field fluorescence imaging. The number of nuclei per field of view was counted in ImageJ using the following image processing steps: ‘Threshold’ (20–30 to 255); ‘Watershed’; and ‘Analyze Particles’ (200–infinity). ASC specks were counted in ImageJ in the GFP channel using ‘Find Maxima’ (prominence = 20).

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