Cellstar 96 well tissue culture plates

Freshly grown Lactobacillus cultures were pelleted at 4500×g for 10 min at 4 °C, washed twice and re-suspended in sterile phosphate-buffered saline (PBS, pH 7.2). Each pellet was diluted to OD_600nm = 0.05 in PBS at pH 1–5, 7 (adjusted with 1.0 N HCl) and incubated at 37 °C for 1, 2, and 4 h. Subsequently, 20 µL of the bacteria were inoculated into Cellstar 96-well tissue culture plates (Greiner Bio-One, Monroe, NC, USA) containing 180 µL of MRS broth and incubated at 37 °C for 24 h. Growth was determined by OD_600nm readings on a microplate reader.

The indicated cells were seeded in CELLSTAR® 96-well tissue culture plates (Greiner Bio-One GmbH) at appropriate densities the day before treatment. For necroptosis induction, cells were pretreated with 1 µM BV6 and 20 µM zVAD.fmk for 30 min (if not stated otherwise), followed by stimulation with 10 ng/mL recombinant human TNFα for the indicated time (TBZ). The inhibitors HOIPIN-8 (30 µM), Gliotoxin (1 µM), Nec-1s (30 µM), GSK’872 (20 µM) and NSA (10 µM) were added 1 h prior to TBZ treatment. Cell death was determined by PI and Hoechst33342 (Hoe) staining and fluorescence-based quantification of dead (PI-positive) cells compared to the total cell number (Hoe-positive cells) using an ImageXpress® Micro XLS Widefield High-Content Analysis System and MetaXpress® Software (Molecular Devices Sunnyvale, CA, USA).