Sera plus

To analyze effects of FAP-IL-2v on the cellular cytotoxic activity of effector cells (ADCC) mediated by DB, a non-radioactive calcein-AM-based cytotoxicity assay was used, as previously described [10 (link)]. Briefly, leukocytes of healthy donors were cultivated for five days using RPMI 1640 (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) supplemented with 2 mM stable glutamine, 0.3× P/S and 10% Sera Plus (PAN-Biotech GmbH, Aidenbach, Germany). To show the effects of FAP-IL-2v on ADCC, culture medium was supplemented with 1 µg/mL/day of FAP-IL-2v. Untreated leukocytes and leukocytes incubated with IL-2 (3000 IU/mL/day) served as controls. To induce ADCC against NB cells, DB (10 µg/mL) and an effector-to-target cell ratio of 40:1 were used. The GD₂-positive human NB cells LAN-1 (5000 cells/well) served as targets cells. The GD₂ specificity of ADCC was confirmed using the anti-idiotype Ab ganglidiomab [13 (link)]. The DB-independent cytotoxicity of leukocytes (AICC, antibody-independent cellular cytotoxicity) was evaluated by incubation of leukocytes with tumor cells with rituximab.

Primary cultures of human dermal fibroblasts were purchased from Istituto Zooprofilattico Sperimentale, (Brescia, Italy). Human dermal fibroblasts were cultured in Minimum Essential Medium (GIBCO) supplemented with 10% fetal bovine serum (SERA PLUS, PAN biotech GmbH), penicillin (100 U mL⁻¹), streptomycin (100 μg mL⁻¹) and L-glutamine (2 mM), and maintained in an Heraeus BB15 incubator (Thermo Scientific, Germany) at 37 °C, 5% CO₂ under humidified atmosphere. Cell culture medium was changed every 2–3 days and fibroblasts were routinely sub-cultured at 80% confluence by trypsinization. For the experiments, cells were seeded at an optimal density of 10 × 10³ cells/cm².

Primary mouse glial cells were isolated and cultured as described in our earlier publication [32 (link)]. Briefly, 3 male and 3 female mice were used in both the WT (n = 6) and the APOB-100 group (n = 6) for each isolation. Forebrains were obtained from 3 or 4-day-old WT and APOB-100 transgenic mice and placed into ice-cold PBS. Meninges were removed and little pieces of cortices were pipetted into 50-ml tubes and then the tissue was mechanically dissociated by using a long and thin needle (21G 4 ¾, Braun, Germany). Isolated cells were plated onto uncoated T25 flasks (Corning Costar Co.) and cultured in low-glucose DMEM (Thermo Fisher Scientific, Waltham, Massachusetts, USA), which contained 10% fetal bovine serum (Sera Plus, Pan Biotech, Aidenbach, Germany) and gentamycin (50 μg/ml). Glial cells were cultured until confluency with medium change every 2 days. Then glial cells were seeded onto poly-l-lysine-coated coverslips placed into 24-well plates and cultured with medium change every 3 days. Cultures reached confluency in 5–7 days. Then they were treated with cytokines, fixed and immunostained. Confluent cell layers consisted of 57% astrocyte and 43% microglia in WT, and 49% astrocyte and 51% microglia in APOB-100 glia cultures.

Human MSCs were obtained from bone fragments of patients undergoing hip replacement surgery as approved by the ethics committee at the Philipps-University Marburg (study no. 64/01 and 25/10). MSCs were isolated and cultivated as described previously [11 (link), 14 (link)]. Dulbecco’s Modified Eagle Medium (DMEM) containing 1 g/l d-glucose (Gibco by Life Technologies, Carlsbad, CA, USA) supplemented with 1% penicillin/streptomycin (P/S (100×) P11-010; PAA Laboratories GmbH, Pasching, Austria) was supplemented with either 10% fetal calf serum (FCS; Sera Plus; PAN Biotech GmbH, Aidenbach, Germany) or 10% human platelet lysate (HPL). Cells were incubated at 37 °C with 5% CO₂. MSCs were passaged when they reached ~80% confluence. The immunophenotype and differentiation potential of MSCs were tested as recommended [18 (link)] and described previously [11 (link)]. MSCs at low passage numbers were frozen in 10% DMSO at –80 °C and stored in liquid nitrogen. Cells were thawed and allowed to equilibrate at least 3 days before being used for further experiments.

Human hepatocellular carcinoma (HepG2) (ATCC, HB-8065) were maintained in DMEM (4.5 g/l glucose, pyruvate) (Gibco), supplemented with 10% fetal bovine serum (PAA Laboratories) and penicillin/streptomycin (25 μg/ml each, Gibco) at 37 °C, 5% CO₂. Cells were seeded at a density of 60,000 cells/cm² and allowed to proliferate for 3 days. Following medium change, cells were treated as indicated. Cells were routinely (6 times per year) tested for mycoplasma contamination.
Primary human hepatocytes (PHH) were obtained from BioIVT (donors IAN, IPH, GID) and from Lonza (donors HUM4108, HUM4055B, HUM4229, HUM181501B). Cells were seeded on collagen-coated plates (250 μg/ml rat collagen, Roche) at a density of 150,000 cells/cm² and cultivated for 3 h in William’s E medium (PAN Biotech) containing penicillin (100 U/ml, Gibco), streptomycin (0.1 mg/ml, Gibco), gentamycin (10 μM, PAN Biotech), dexamethasone (100 nM, Sigma), stable l-glutamine (2 mM, Sigma), insulin supplement (2 ng/ml, Sigma), and 10% Sera Plus (PAN Biotech). After 3 h for cell attachment, medium was exchanged to Sera Plus-free medium and cells were allowed to adjust for 16 h before treatment.

The gastric cancer cell line Hs746T (ATCC) was cultured at 37°C in humidified atmosphere with 5% CO₂. Cells were grown in Dulbecco’s Modified Eagle Medium with GlutaMAX (Thermo Fisher Scientific) with 0.5% penicillin/streptomycin (Thermo Fisher Scientific) and 10% Sera Plus (Pan Biotech). The absence of mycoplasma was tested as described elsewhere [5 (link)].