*4.3. Screening of Necroptosis Inhibitors*

BV2 cells were plated in 96-well plates at 7 × 103 cells/well and after 24 h, necroptosis was induced by adding 25 μM zVAD-fmk in RPMI/ITS. Test compounds or Nec-1 (positive control of necroptosis inhibition) were incubated along with zVAD-fmk at a final concentration of 30 μM for 24 h. DMSO was used as vehicle control. Cell viability was determined based on measurement of MTS metabolism using the CellTiter 96® Aqueous Non-Radioactive Cell Proliferation (MTS) Assay (Promega, Madison, WI, USA). Differences in absorbance were measured at 490 nm using GloMax® Multi Detection System (Sunnyvale, CA, USA).
