Yo pro 1

LDH release was measured in HeLa cells transfected with human RIPK1 (wild type and mutant) to determine necroptosis using the Cytotoxicity Detection Kit (Roche, Switzerland) following the manufacturer’s instructions. The results were expressed as the percentage of total LDH intracellularly present in control cells. Necroptosis was also followed by Yo-Pro-1 uptake, for that HeLa cells transfected with the different RIPK1 plasmids were incubated with 2.5 μM Yo-Pro-1 (Invitrogen, USA) for 5 min, and then fluorescence was determined at 485 ± 9/515 ± 9 nm (excitation/emission) in a Synergy Mx plate reader (BioTek, USA), and the percentage of Yo-Pro-1 was compared to cells treated with 0.1% triton X-100 for 5 min.

The presence of lactate dehydrogenase (LDH) in cell culture supernatants was measured using the Cytotoxicity Detection kit (Roche), following manufacturer’s instructions. It was expressed as the percentage of the total amount of LDH present in the cells. For Yo-Pro uptake, macrophages were preincubated for 5 min at 37 °C with 2.5 µM of Yo-Pro-1 iodide (Life Technologies) after galvanic current application or 1% triton X100 (Sigma-Aldrich) application. Yo-Pro-1 fluorescence was measured after the treatments every 5 min for the first 30 min and then every 30 min for the following 3 h with an excitation wavelength of 478 ± 20 nm and emission of 519 ± 20 nm in a Synergy neo2 multi-mode plate reader (BioTek). Intracellular K⁺ was quantified using macrophages lysates, as has already been reported (Compan et al., 2012 (link)), and measured by indirect potentiometry on a Cobas 6000 with ISE module (Roche).

Cells were incubated with 2.5 μM Yo-Pro-1 (Life Technologies) for 5 min, and fluorescence was recorded every 10 s for 35 min at 37 ºC, before and after the addition of melittin or nigericin at different concentrations as annotated in the figure legends. Fluorescence of DNA-bound Yo-Pro-1 was measured in a Synergy Mx plate reader (BioTek) at 485±9 nm excitation and 515±9 nm emission.