Albumin fraction 5

The collected BMOs were fixed with 2% paraformaldehyde (PFA, ThermoFischer Scientific, 15434389) for 1 h and permeabilized with 0.3% Triton-X-100 (Sigma, T8787) in PBS for 3 h at RT. Depending on the secondary antibody either 1% Albumin Fraction V (AppliChem, A1391.0500) or 10% donkey-serum in PBS 0.01% Triton-X-100 as blocking solution was used at 4 °C overnight. After the blocking step, the following primary antibodies were used for the immunostainings (supplementary material Table 2) in combination with the below listed secondary antibodies (supplementary material Table 3). DAPI was used to mark the nuclei.

Sections on the wafers were washed with PBS (0.1 M, pH 7.4, 4 °C) for 20 min followed by incubation with 0.15% glycine in PBS (3 × 1 min) and washes in PBS (3 × 10 sec). Blocking solution composed of 0.5% BSA (Albumin Fraction V, Applichem) and 0.2% gelatin (from bovine skin, Sigma) in PBS was applied for 5 min. Primary antibodies, anti-NuP complex, (4 μg/ml, mAB414, Covance), anti-Tom20 (1 μg/ml, FL-145, Santa Cruz) and pNCC (1:250, antibody kindly provided by Prof. Johannes Loffing, University of Zurich, Switzerland) dissolved in blocking solution were incubated for 40 min at room temperature. After washes with blocking solution (6 × 20 sec) wafers were incubated with secondary antibodies (anti-mouse Alexa Fluor 568, 8 μg/ml, Life Technologies; anti-rabbit Alexa Fluor 488, 8 μg/ml, Life Technologies; anti-rabbit Alexa Fluor 647 F(ab’)₂, 6 μg/ml Jackson Immuno-Research for GSDIM experiments) in blocking solution (40 min at room temperature). For the immunogold experiments, 12 nm gold conjugated goat anti-rabbit antibodies were incubated for 40 min at room temperature (Optical density OD₅₂₅ 0.15). Washes in blocking solution (6 × 2 min) were followed by PBS (3 × 2 min) and post-fixation with 0.025% glutaraldehyde in PBS for 5 min. Finally, the wafers were transferred to a 6 well plate filled with PBS. Sections were stored at 4 °C until further processing.

5*10⁵ HeLa cells were stimulated for indicated time periods with CD95L [33 (link)]. When indicated, cells were pre-stimulated with 50 μM zVAD-FMK (Bachem AG, Bubendorf, Switzerland) or 10 μM IKK inhibitor VII (MerckMillipore) for 30 minutes. After stimulation medium was removed, cells were washed with PBS and detached with trypsin-EDTA solution (Life Technologies, Darmstadt, Germany) for 5 minutes at 37°C. Afterwards, cells including medium, washing PBS, and trypsin were spun down at 500xg at 4°C for 5 minutes. Cell were fixed with 3% formaldehyde in PBS for 10 minutes at room temperature, permeabilized with 90% ice-cold methanol for 30 minutes on ice and washed twice with incubation buffer (5 g/l albumin fraction V (AppliChem, Darmstadt, Germany) in PBS). For antibody staining, cells were resuspended in 50 μl incubation buffer. Cells were stained for one hour in the dark with 1 μl anti-caspase-3 antibody, recognizing caspase-3 cleaved at Asp175, conjugated to AlexaFluor488 or AlexaFluor647, 0.5 μl anti-NF-κB-p65 antibody, conjugated to phycoerythrin (PE), (both purchased from Cell Signaling Technologies). After staining, cells were washed with incubation buffer and resuspended in 40 μl PBS. At least 5 minutes before measurement, 3 μl of the DNA dye 7AAD (BioLegend, San Diego, USA) was added.