L929 fibroblast cell line derived from mouse adipose tissue was obtained from Korean Cell Line Bank (Seoul, Korea). L929 cells were cultured in RPMI1640 media supplemented with L-glutamine (300 mg/L), 25 mM HEPES, 25 mM NaHCO3, and 10% FBS. To induce necroptosis, cells were treated with a mixture of 30 ng/mL TNFα (PeproTech, Rocky Hill, NJ), 10 μM LCL-161 (R&D Systems, Minneapolis, MN, USA), and 20 μM Z-VAD-FMK (R&D Systems) for 40 min [28]. Cells were fixed and subjected to immunofluorescence staining with anti-pMLKL and anti-pRIPK1 antibodies to establish specificity of these antibodies.
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