Gentamicin

Progenitor-derived astrocyte (PDAs) were generated from neural progenitor cells (kindly provided by Dr. Eugene Major, NINDS, NIH, MD) as previously described [14 (link)]. Briefly, PDAs were maintained in progenitor medium consisting of Neurobasal media (Invitrogen) supplemented with 0.5 % bovine albumin (Sigma-Aldrich), neurosurvival factor (NSF; Lonza), N2 components (Invitrogen), 25 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml EGF (R&D Systems), 50 μg/ml gentamicin (Lonza), and 2 mM L-glutamine (Invitrogen). To induce differentiation, progenitor medium was replaced with PDA medium containing DMEM supplemented with 10 % heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 50 μg/ml gentamicin. Cultures were >90 % positive for glial fibrillary acidic protein (GFAP) after 30 days of differentiation. Fetal-derived Normal Human Astrocytes (NHAs, Lonza) were maintained in Astrocyte Growth Media (AGM) BulletKit (Lonza) supplemented with 0.3 % heat-inactivated fetal bovine serum, 1 ml/ml ascorbic acid, 1 ml/ml rhEGF, 1 ml/ml AG-1000 (30 mg/ml gentamicin and 15 μg/ml amphotericin), 2.5 ml/ml insulin, and 10 ml/ml L-glutamine. Adherent primary cells were removed by treatment with 1 mM EDTA for 5 min with gentle scraping or pipetting multiple times.

Mouse Foxo1 cDNA was cloned into an MSCV-IRES2-EGFP retroviral vector. Retrovirus was made by transfection of HEK-293T cells. BM cells were collected from the femurs and tibiae of 6- to 8-week-old donor mice. After red blood cell lysis, hematopoietic stem cells were enriched using the Lineage Cell Depletion Kit (Miltenyi Biotec). Hematopoietic stem cells were cultured in chemically defined serum-free medium X-VIVO 10 with gentamicin (Lonza) supplemented with L-glutamine, β-mercaptoethanol (50 mM), mouse recombinant stem cell factor (50 ng/mL), IL-6 (20 ng/mL), IL-3 (10 ng/mL), FLT-3L (10 ng/mL), and IL-7 (10 ng/mL) (PeproTech) for 24 hours. Then, cocultured cells were transduced by spin infection with retroviral supernatant. Cells were incubated for another 24 hours before intravenous tail injection into WT recipient mice, which were irradiated at 9 Gy for at least 12 hours before adoptive transfer. 6 weeks after transfer, chimeras were analyzed by flow cytometry.

Neutrophils and macrophages were cultured in 24-well flat-bottom cell culture plates at 5 × 10⁵/well in IMDM medium (Lonza) supplemented with 5% fetal bovine serum (FBS; Lonza), 2 mM stable l-glutamine (Cytogen), and 50 mg/ml gentamicin (KRKA) at 37 °C in an atmosphere of 5% CO₂. To determine the influence of the biofilm forms of P. aeruginosa on innate immune cell activity, neutrophils and macrophages were stimulated with heat-killed whole bacterial cells (PAR5-72 h 20:1 and 100:1 bacteria per cell) or with 72-h conditioned media from bacterial cultures (BCM-72 h at 5–20% total volume), if not otherwise stated. The effect was compared with that of bacterial cells (PAR5-8 h) and 8-h conditioned media (BCM-8 h). As a reference stimulus for N1/M1 neutrophils/macrophages, we used 100 ng/ml LPS from Escherichia coli strain 0111:B4 (LPS, Sigma-Aldrich). After 24 h of stimulation, culture supernatants were collected and frozen at − 80 °C until use. Cells were used in a western blot analysis. In some experiments, neutrophils were cultured with BCM-72 h treated with inhibitors of DNA (BCM were incubated in the presence of 2.5 µl of 1500 Kunitz DNAse I, Qiagen; chloroquine, Sigma-Aldrich—2.5 µg/ml) or/and with the inhibitor of LPS (polymyxin B, Sigma-Aldrich, 100 µg/ml) or preincubated with EPS (30 µg/ml) for 1 h and then re-stimulated with LPS (100 ng/ml) or DNA (3 µg/ml).

Human gingival mesenchymal stem cells (hGMSCs) were isolated from the gingival tissues of three patients, in good general health conditions and without oral disease, who underwent a surgical procedure. The gingival tissues were placed in a culture dish, fragmented, washed several times in phosphate-buffered saline solution (PBS, Lonza, Basel, Switzerland) with 5% of gentamicin (Lonza) and transferred into the incubator at 37 °C in a humidified atmosphere of 5% CO₂ in air with mesenchymal stem cell growth medium-chemically defined (MSCGM-CD, Lonza). The medium was replaced every two days for approximately two weeks before the cells reached 80% confluence and were passaged in culture [26 (link)].

For the autologous tumor lysate preparation, a cervical punch biopsy was collected in 10 mL of Hank’s balanced salt solution (Lonza, Switzerland) (ice cold) containing 100 IU per mLs of penicillin, streptomycin, and gentamicin (Lonza, Switzerland) each. Sample processing was carried out on the same day of collection and processed in 6 mL of HBSS containing 0.6 Pz units of collagenase (NB6 GMP-grade from Serva GmBH, Heidelberg, Germany) and incubated overnight at 37 °C/5% CO₂ to allow for tissue dissociation). Traces of collagenase were removed by washing with calcium-free DPBS. The final cell suspension was passed through a sterile 70 μm cell strainer (Miltenyi MACS, Waltham, MA, USA) to obtain a fine single-cell suspension. Approximately 2 × 10⁵ cells were immobilized on slides and verified by a cytologist to obtain the tumor cell percentage. The remaining cells in the single-cell suspension (SCS) were frozen in a ready-to-use, serum-free, GMP-grade cryopreservation medium with 10% Dimethyl sulfoxide (DMSO) (CTS™ Synth-a-Freeze™ Medium—Gibco™, Waltham, MA, USA) as per the manufacturer’s instructions. The tumor lysates were prepared as described previously [19 (link)] and evaluated for sterility followed by cryopreservation at −80 °C until use for antigen priming.