Fbs superior

To confirm the purity of primarily isolated EVT cells FACS analysis was performed. For intracellular trophoblast-specific CK 7 staining, permeabilization-buffer was made from 0.1% saponin (Carl Roth, Karlsruhe, Germany), 5% FBS superior (Sigma-Aldrich, St. Louis, MO, USA), and D-PBS (Life Technologies, Carlsbad, CA, USA). The Fc receptors were blocked in 10% human serum in D-PBS for 10 min at RT. For the surface staining, the cells were incubated in FACS-staining-buffer with CD45-FITC (BioLegend, San Diego, CA, USA) for 15 min at 4 °C. Then the cells were stained with fixable live/dead dye eFluor780 (Thermofisher, Waltham, MA, USA). Next, they were fixed with 1% PFA for 10 min at RT. For intracellular blocking, the cells were resuspended in permeabilization-buffer and 10% human sera was added for 10 min at 4 °C. Intracellular staining was executed with a CK 7-PE antibody (Abcam, Cambridge, UK) diluted in permeabilization-buffer. Before acquisition, the cells were washed in permeabilization-buffer and FACS-staining-buffer. Flow cytometry was performed on BD FACSCanto II and analyzed with FlowJo version 10.

Human liver hepatoma (HepG2) cells were obtained from American Type Culture Collection (ATCC HB-8065, Manassas, VA, USA) and were cultivated in Iscove’s modified Dulbecco medium (IMDM, Lonza) supplemented with 10% fetal calf serum (FBS Superior, Sigma-Aldrich) at 37 °C and 5% CO₂. A WST-1 (Roche, Mannheim, Germany) assay was conducted to quantify cell proliferation. To this end, 2 × 10⁴ cells were seeded in 200 µL IMDM supplemented with 10% FCS in 96-well cell culture plates (Greiner-CELLSTAR, Sigma-Aldrich) and incubated for 24 h (37 °C, 5% CO₂). Afterwards, medium was replaced by fresh medium containing 10 to 100 µM of buprestin H in ethanol. Ethanol-treated cells served as control. After another 48 h of cultivation, medium was again removed and 110 µL of 10% WST-1 reagent in phosphate-buffered saline (PBS, Gibco, Thermo Fisher Scientific) was added. Absorbance was measured at 450 nm after 2 h by using a Tecan Safire II (n = 4 for every concentration).

Mammalian cells were cultured in DMEM (Gibco) + GlutaMAX (Thermo Fisher Scientific) supplemented with 10% FBS (FBS Superior; Sigma-Aldrich), 1 mM sodium pyruvate (Thermo Fisher Scientific), and Penicillin-Streptomycin (100 µl/ml and 0.1 mg/ml; Sigma-Aldrich) at 37 °C and 5% CO₂. Peroxisomes in Vero cells were labeled as described^{4 (link)}. In brief, cells were fixed in 8% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), permeabilized (0.5% Triton X-100/PBS), and blocked (2% BSA/0.1% Tween20/PBS). Then cells were incubated for 1 h using antibodies against PMP70 (1:200; abcam, code: ab3421). Primary antibodies were detected using secondary Alexa Fluor 647 AffiniPure Donkey Anti-Rabbit IgG (H + L) antibodies (1:400; Jackson ImmunoResearch, 711-605-152). Secondary antibodies were as well incubated for 1 h. Samples from neurons were prepared using primary antibodies against betaII spectrin (BD Biosciences, 612563) as described^{32 (link)}. In brief, cultures of hippocampal neurons were prepared` from Wistar rats of mixed sex at the 1st postnatal day. After 20–30 days of cultivation on coated coverslips, cells were washed in PBS and fixed (4% PFA/PBS). After quenching the PFA (100 mM NH₄Cl and 100 mM Glycine), permeabilization (0.1 % Triton X-100), and blocking (1% BSA/PBS), primary and secondary antibody incubations were performed (in PBS) for 1 h at room temperature.

According to standard protocols W4 murine embryonic stem cells (mESCs), originally isolated from the 129S6 mouse strain, were grown as described previously [9 (link)]. In brief, cells were cultured in DMEM supplemented with 15% FBS Superior (Biochrom AG, Berlin, Germany), 1% Cell Shield® (Minerva Biolabs GmbH, Berlin, Germany), 100 µM non-essential amino acids, 1000 U/mL leukemia inhibitory factor (Phoenix Europe GmbH, Mannheim, Germany) and 100 µM β-mercaptoethanol (Sigma-Aldrich GmbH, Steinheim, Germany) at 37 °C, 5% CO₂, and 20% O₂. We performed cardiovascular differentiation using cardiogenic differentiation medium, containing IBM (Iscove’s Basal Medium, Biochrom AG, Berlin, Germany) supplemented with 10% FBS Superior, 1% Cell Shield®, 100 µM non-essential amino acids, 450 µm 1-thioglycerol (Sigma-Aldrich GmbH, Steinheim, Germany), and 213 µg/mL ascorbic acid (Sigma-Aldrich GmbH, Steinheim, Germany). Cardiovascular differentiation was initiated by hanging-drop culture for two days at 37 °C, 5% CO₂, and 20% O₂. To start formation of embryoid bodies (EB) 400 cells per drop were plated on the cover of a square petri dish and grown for 2 days. Subsequently, EB grew for four more days in suspension culture and were then harvested for transplantation. These cells will be referred to as cardiac induced cells (CiC) [8 (link)].

FaDu (hypopharyngeal carcinoma) and Kyse30 cells (esophageal squamous cell carcinoma) used in this study were purchased from DSMZ (Braunschweig, Germany). Peritumoral fibroblasts (PtFs) were isolated from HNSCC patient biopsies from mucosa macroscopically free of cancer (35 (link)).
To create red fluorescent FaDu and Kyse30 cells, parental cells were transfected with the pCAG-ef1-mCherry plasmid and stable cell lines were created by drug selection with 1 µg/ml puromycin.
Cells were grown in T25 or T27 cell culture treated flasks (Sarstedt AG, Nümbrecht, Germany) in an incubator at 37°C, 5% CO₂ and 100% humidity, and passaged 1:5 to 1:20 (FaDu, Kyse30) or 1:4 (NF8) two to three times weekly. Kyse30 cells were grown in RPMI 1640 with L-Glutamine (Gibco, Dublin, Ireland) supplemented with 10% FCS and 1% Penicillin/Streptomycin (final concentration: 100 µg/ml, Gibco, Dublin, Ireland). For FaDu cells, DMEM (Gibco, Dublin, Ireland) supplemented with 10% FCS (FBS Superior, Sigma Aldrich, St.Louis, USA) and 1% Penicillin/Streptomycin (final concentration: 100 µg/ml, Gibco, Dublin, Ireland) was used. NF8 cells were grown in Fibroblast Growth Medium 2 (FGM-2) (C-23220, PromoCell, Heidelberg, Germany) supplemented with Supplement Mix 2 (C-39325, PromoCell).

The pre-B cell lines 1676, wk3, and TKO were derived by extended culture of total bone marrow of SLP-65^−/−LAT^−/−, SLP-65^−/−, or Igll1^−/−Rag2^−/−SLP-65^−/− (TKO) mice, respectively, in IL-7–supplemented medium and have been previously described (23 (link), 65 (link)). Pre-B cell lines, primary bone marrow–derived pre-B cells, and 293T cells were cultured in IMDM (Sigma-Aldrich) containing 7.5% FBS (Superior; Sigma-Aldrich), 100 U/ml penicillin, 100 U/ml streptomycin (Sigma-Aldrich), and 50 μM 2-ME. For pre-B cells, this medium was supplemented with the supernatant of IL-7–expressing J558L cells in excess if not stated otherwise. The Syk kinase was inhibited using compound R406 (Selleckchem) at a concentration of 2 μM. For analysis of DNA damage induced apoptosis, cells were treated with 5 nM Etoposide for 20 h.

The AML cell lines TF-1 and OCI-AML3 as well as the NK-92 were purchased from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany), and the MV-4-11 cell line was purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). MV-4-11 cells were cultured in RPMI 1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and supplemented with 10% fetal bovine serum (FBS superior, Sigma-Aldrich, St. Louis, MO, USA). TF-1 cells were cultured in RPMI 1640 supplemented with 20% FBS and 5ng/mL human granulocyte-macrophage colony-stimulating factor (GM-CSF, PeproTech GmbH, Hamburg, Germany). OCI-AML3 cells were cultured in MEM-Alpha medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 20% FBS. NK-92 cells were cultured in MEM-Alpha medium supplemented with 12.5% FBS, 12.5% horse serum (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL Interleukin 2 (Recombinant human Interleukin-2, PeproTech GmbH, Hamburg, Germany), and 0.1 mM 2-Mercaptoethanol (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The NK-92 cells express TIGIT and CD39, which were regularly assessed by MFC. Cell cultures were incubated at 37 °C and 5% CO₂.