Iscove modified dulbecco media (imdm)

Murine malignant melanoma B16-F10 cells (ATCC CRL-6475) were grown in Iscove’s Modified Dulbecco’s Medium (IMDM; P04-20350; Panbiotech; Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS; 26140079; Fisher Scientific; Waltham, MA, USA) and antibiotics. Cells were incubated under 37 °C with an atmosphere of 5% CO₂. B16-F10 cells were treated at a final concentration of 2 μg/mL of DOX-loaded SLPs (SLPs-DOX) for 2, 16, or 48 h. For confocal fluorescence imaging purposes, cells were fixed with 4% paraformaldehyde (43368; Alfa Aesar; Haverhill, MA, USA) and were immunolabeled with the anti-α-tubulin (B512; Sigma; Germany) antibody to recognize the microtubules. A secondary goat anti-mouse 647 (A21236; Invitrogen; USA) was used. Fluorescent images were obtained with a Nikon A1R confocal microscope (Tokyo, Japan). All images were pseudocolored.

We generated lentiviral particles by transfecting 6 × 10⁶ HEK293 T cells with 6 μg of each of the following plasmid DNAs, as described in detail previously [38 (link)]): (I) packaging vectors pczVSV-G [40 (link)] and pCD NL-BH [40 (link)], (II) pLenti_CMV_INPP4B for transduction of etoposide resistant Y79 and RB355 cells, (III) pLenti_CMV_Puro_Dest as a negative control for all overexpression experiments or (IV) GFP expression vector (pCL7EGwo) each together with 45 μg polyethyleneimine (PEI, branched, Sigma-Aldrich, St. Louis, Missouri, USA) in DMEM medium. The medium was changed to Iscove's Modified Dulbecco's medium (IMDM, Pan-Biotech, Aidenbach, Germany) with 10% FCS and 1% penicillin/streptomycin after 24 h. 72 h later we harvested the viral supernatants, filtered and cryoconserved them.
For stable transduction, 1 × 10⁶/0.8 × 10⁶ RB cells (RB355/Y79) were seeded in DMEM medium as described previously [41 (link)]. The medium was removed after 24 h, and cells were transfected with INPP4B virus or control virus particles, and 5 μL polybrene (H9268, Sigma-Aldrich, München, Germany) per ml lentivirus were administered. Twice the volume of the virus particles DMEM medium with supplements was added after 24 h. Forty-eight hours later we changed the medium completely and incubated the cells for another 72 h.

HEK293T cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Corning), 10,000 U/mL penicillin, 10 mg/mL streptomycin, and 2 mM L-glutamine.
Primary murine BMDCs were obtained from tibia and femur bone marrows of C57BL/6 mice and cultured in Iscove’s modified Dulbecco’s medium (IMDM, PAN-Biotech) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% granulocyte-macrophage colony stimulating factor (GM-CSF) containing supernatant derived from X63 cells (72 (link)). Medium was exchanged every 48 hours and BMDCs were used after 8 days to ascertain that ≥80% of the cell population expressed the DC differentiation marker CD11c.
Primary human moDCs were obtained from PBMCs isolated from blood of healthy donors via Ficoll density gradient centrifugation. CD14⁺ monocytes were isolated by magnetic-activated cell sorting (Miltenyi Biotech) and differentiated into moDCs for 5 days in serum-free CellGenix GMP dendritic cell medium (CellGenix) supplemented with 1,000 U/mL GM-CSF (Miltenyi Biotech) and 1,000 U/mL interleukin-4 (IL-4, Miltenyi Biotech). All cells were grown at 37°C and 5% CO₂ in humidified atmosphere.

Isolated PBMC were seeded into sterile 96-well flat-bottom plates at 10⁶ cells/well in 200 µL Iscove’s Modified Dulbecco’s Medium (IMDM; PAN-Biotech GmbH, Aidenbach, BY, Germany) supplemented with 10% fetal bovine serum (FBS, PAN-Biotech GmbH, Aidenbach, BY, Germany), 50 µg/mL gentamicin (Merck KGaA, Darmstadt, BW, Germany), 100 U/mL penicillin, and 100 µg/mL streptomycin (Pen/Strep; Merck). Viable S. suis strains from glycerol stocks were added at PBMC-to-bacteria ratios of 10:1 or 1:1 (corresponding to 10⁵ and 10⁶ cfu/well), as indicated in the respective figure descriptions and legends.
For the separation experiments, PBMC were again cultivated at 10⁶ cells/well (200 µL). CD14-positive monocytes were cultivated at 5 × 10⁴ cells/well (5% of PBMC); granulocytes were cultivated at 1 × 10⁶ cells/well. For stimulation, the same final concentration of 10⁶ cfu/well was used for all three fractions (equivalent to a 1:1 ratio of PBMC to bacteria).
For IL-10 neutralization, anti-porcine IL-10 (clone 148801; R&D Systems Inc.) or isotype control mIgG2b (clone 20116; R&D Systems Inc.) were added at 0.1 µg/mL, respectively. Bioactivity of clone 148801 has been demonstrated previously [56 (link)]. After 42 h of stimulation with S. suis, supernatants were taken and stored at −20 °C.

BMCs were collected from femurs and tibias of adult rats, flushed with sterile phosphate-buffered saline (PBS; Carl Roth GmbH, Karlsruhe, Germany), incubated in red blood cell lysis buffer (Sigma, Welwyn Garden City, UK) and cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, Pan Biotech, Aidenbach, Germany) supplemented with 20% Fetal Bovine Serum (FBS, Biochrom AG, Berlin, Germany), 100 IU mL⁻¹ penicillin and 100 mg mL⁻¹ streptomycin (Corning, Corning, NY, USA). The initial culture medium was changed at passage 1 to Dulbecco’s Modified Eagle’s Medium–high glucose (DMEM, Sigma–Aldrich, Schaffhausen, Switzerland), with 10% FBS, 100 IU mL⁻¹ penicillin and 100 mg mL⁻¹ streptomycin. Cells were cultured under aseptic conditions using sterile and RNAse/DNAse free tissue culture-treated plastic ware (Corning, NY, USA) at 37 °C and 5% CO₂ in a humidified incubator (Model CB160, Binder, Tuttlingen, Germany). The medium was replaced every two days, and upon 80% confluence, cells were trypsinized, pooled, and subcultured until passage 2. In order to preserve the different populations of the BMCs, cells were collected at passage 2, and no other selection was performed.
For in vivo study, each pool was obtained from 10 male Lewis rats (mean weight of 150 g). For the in vitro study, each pool of BMCs was prepared from 3 male Lewis rats (mean weight of 150 g).

Cell lines 293FT, A549, 1321 N1, Shh-Light2 (Shh-L2) and U2OS were purchased from ATCC. HUH7 cells were a kind gift from Dr. Pirjo Spuul. Adult pooled normal human epithelial keratinocytes (NHEKs, PromoCell) (kind gift from Dr. Ana Rebane) were grown in Defined Keratinocyte-SFM Medium (DKSM) (Gibco, Thermo Fisher Scientific). 293FT, A549, 1321 N1, and Shh-L2 cell lines were cultured in Dulbecco's Modified Eagle Medium-high glycose (DMEM, Pan Biotech) supplemented with 10% foetal calf serum (FCS, Pan Biotech) and 1% penicillin/streptomycin (PEST, Sigma-Aldrich). The growth medium of Shh-L2 cells also contained 0.4 mg/ml G418 (Sigma-Aldrich) and 0.1 mg/ml zeocine (Invitrogen). U2OS cells were propagated in Iscove's Modified Dulbecco's Medium (IMDM, Pan Biotech), 10% FCS and 1% PEST. Cells were propagated at 37°C in 5% CO₂. HUH7 cells were transfected using Lipofectamine 2000 (Life Technologies). 293FT and Shh-L2 cells were transfected using polyethylenimine (PEI) as previously described [9 (link)]. U2OS cells were co-transfected with HPV minicircle genomes and GLI1FL or GLI1ΔN encoding constructs by electroporation (220 V and 975 μF) using a Gene Pulser XCell system (Bio-Rad Laboratories).

OCI-Ly1and OCI-Ly8 cells were cultured in IMDM (PAN Biotech, Aidenbach, Germany). 293 T HEK cells and HeLa cells were cultured in Dulbecco’s Modified Eagle Medium. All cells were cultured with 10% FBS (PAN), 37 °C 5% CO₂. Cell lines were authenticated by short tandem repeat analysis (Eurofins, Val Fleuri, Luxembourg) and tested negative for mycoplasma by PCR. Of note, we confirmed that OCI-Ly1 harbors a variant (G375R) in the STAT6 DNA-binding domain [23 (link)] that, to the best of our knowledge, has not been reported in any other cell line or primary tumor sample. Cells were stably transduced with a CMV-driven cDNA expression construct (pHAGE-CMV-MCS-IRES-ZsGreen; PlasmID, EvNO00061605) encoding for Flag-tagged mutant (MUT) STAT6 (D419G, D419N, N421K, or D519V) or wild type (WT) STAT6 (PlasmID, HsCD00365550) as previously described [7 (link)], and stimulated with human recombinant IL-4 (Miltenyi Biotec, Cologne, Germany) as indicated.