Mercaptoethanol

Liver nonparenchymal cells (NPC) were isolated using a liver dissociation kit and the gentle MACS dissociator (both Miltenyi Biotec) as recommended by the manufacturer with some modifications. In brief, after liver dissociation, cells were resuspended in 1 mL cold HBSS buffer (w/o Ca²⁺ and Mg²⁺). The cell suspension was mixed with a double volume of freshly prepared 30% HistoDenZ (Sigma-Aldrich) and overlaid with 1 mL of cold HBSS buffer. After centrifugation (1500× g, 4 °C, w/o break), the cell containing interphase was retrieved and washed. Erythrocytes were lysed using a hypotonic buffer.
Spleen cells were isolated using a 40 µM cell strainer (Greiner Bio-One) to obtain a single-cell suspension. In parallel settings, spleen cells were resuspended in medium (IMDM, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin (all from Sigma-Aldrich, Deisenhofen, Germany), and 50 µM ß-mercaptoethanol (Roth, Karlsruhe, Germany)) containing 5% FCS (PAN).
Bone marrow cells (2 × 10⁵/mL) were seeded in 12-well cell cluster plates (Greiner Bio-One) in an IMDM-based culture medium (see above) supplemented with 10 ng/mL recombinant murine GM-CSF (R&D Systems, Wiesbaden, Germany). Culture media was replenished on days 3 and 6 of culture.

The human RB cell lines Rbl13 and Rbl30 were kindly provided by Dr. H. Stephan. The cell lines were cultivated as suspension cultures in Dulbecco’s modified Eagle’s medium (DMEM; PAN-Biotech, Aidenbach, Germany) with 15% fetal calf serum (FCS; PAN-Biotech, Aidenbach, Germany), 100 U penicillin/mL and 100 µg streptomycin/mL (Invitrogen, Darmstadt, Germany), 4 mM L-glutamine (Sigma-Aldrich, München, Germany), 50 µM ß-mercaptoethanol (Carl-Roth, Karlsruhe, Germany), and 10 µg insulin/mL (Sigma-Aldrich, München, Germany) at 37 °C, 10% CO₂, and 95% humidity as described previously (Busch et al. 2015).
The primary RB tumor material was initially cut into small pieces with a sterile scalpel and subsequently washed three times in PBS with a centrifugation step in between (800 rpm for 2 min). After the last washing step, the tumor material was cultivated under the cell culture conditions described above. Cell culture supernatants were harvested and residual cells were removed by centrifugation. Cell culture supernatants were kept at −20 °C until usage.

The following antibodies were purchased from BioLegend (San Diego, CA, USA): Alexa Fluor^TM 647 αHisTag Antibody (Cat. No.: 652513) and Purified anti-HisTag Antibody (Cat. No.: 652501). Peroxidase AffiniPure Donkey αMouse IgG (H + L) antibody (Cat. No.: 715-035-151) was bought from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).
Roswell Park Memorial Institute 1640 medium (RPMI 1640), fetal bovine serum (FBS), L-Glutamine, HEPES, and 0.05% Trypsin-EDTA (1×) were commercially bought from Thermo Fisher Scientific (Waltham, MA, USA). DMSO, Isopropyl-β-D-thiogalactopyranoside (IPTG), penicillin/streptomycin, ß-mercaptoethanol, ampicillin, and lysogeny broth (LB)-medium were purchased from Carl Roth GmbH (Karlsruhe, Germany). PBS, Pancoll, and Minimum Essential Medium (MEM) Non-Essential Amino Acid Solution (100×) were obtained from PAN Biotech (Aidenbach, Germany). Dulbecco´s Modified Eagle´s Medium (DMEM) was provided by Capricorn Scientific GmbH (Ebsdorfergrund, Germany). The DMEM was already supplied with 1.0 g/L L-glucose, 1.0 g/L L-Glutamine, 1.0 g/L sodium pyruvate, and 3.7 g/L NaHCO₃ D-Luciferin Firefly was obtained from Biosynth (Staad, Switzerland).