Dulbecco modified eagle medium (dmem)

Rat BM-MSCs were isolated from the tibias and femurs of donner male rats under sterile aseptic condition. Briefly, after euthanasia, long bones were dissected free of soft tissues, shaved by surgical blades, and cut open at both ends with scissors. Then, bone marrow was harvested by flushing out from the bone cavity with DMEM (Lonza, Basel, Switzerland) using a 22-gauge syringe. Bone marrow was vigorously pipetted repeatedly to produce a single-cell suspension, followed by filtration through a 70 μm nylon mesh filter. After being centrifuged at 1800 rpm for 10 min, the supernatant of the single-cell suspension was discarded. Cell pellets were suspended with complete media and cultured in T25 flasks (CELLSTAR-Greiner bio-one) containing complete media; composed of 89% DMEM, 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin (PS) (1:1) (Lonza, Verviers, Belgium). The medium was refreshed every 2–3 days. All cultures were incubated at 37 °C in a 5% CO₂ under humidified environment. The non-adherent cells were removed by changing the culture medium. When the adherent cells grew to 85–95% confluence, the culture was trypsinized by 0.25% trypsin/EDTA (Gibco, Grand Island, NY, USA) and expanded through three passages (P1–P2). Cells of P2 were used for characterization and transplantation [53 (link)].

HepG2 cells were seeded in 24-well plates. The next day, cells were transduced with adenovirus-GFP (AV-Gfp) or adenovirus-mHilpda (AV-Hilpda) at 5 × 10⁶ IFU/mL media in DMEM (Lonza, Verviers, Belgium) supplemented with 10% fetal calf serum (Lonza) and 1% penicillin/streptomycin (Lonza), hereafter referred to as complete DMEM, and left overnight. Recombinant adenoviruses were generated by cloning GFP or mouse Hilpda cDNA in human adenovirus type5 (dE1/E3). Expression was regulated by CMV promoter. Viruses were produced and titrated by Vector Biolabs (Philadelphia, PA, USA). Cells were then incubated with DMEM 3% BSA and a mixture of oleate and palmitate (ratio 2:1, total concentration 1 mM) for 3 h. Cells were washed twice with PBS and frozen in 25 mM of Tris/HCl and 1 mM of EDTA, pH 7.5. TG quantification plates were thawed, and a mixture of 4:1 tertiary butanol:methanol was added to cells, followed by incubation of 10 min on a shaking platform. Plates were left to evaporate on a hot plate at 50 °C. Next, 300 μL of Triglyceride LiquiColor reagent (Human Diagnostics, Wiesbaden, Germany) was added to the cells and incubated for 10 min while shaking. One hundred microliters were transferred to a 96-well plate, and absorption was measured at 492 nm. A calibration curve of a standard solution was used to determine the TG content of the cells.

Fibroblast BJ cells (BJ, ATCC, Manassas, VA, USA) transduced with lentiviral constructs (exons 1–4) encoding for soluble (s)HLA-B*35:01 or (s)HLA-B*35:08 molecules were maintained in DMEM (Lonza, Basel, Switzerland) supplemented with 20% Medium 199 (Thermo Fisher Scientific Inc., Waltham, MA USA), 10% heat-inactivated fetal bovine serum (FBS; Lonza, Basel, Switzerland), and 2 mM L-glutamine (c-c Pro, Oberdorla, Germany). HEK293T cells were cultured in DMEM (Lonza, Basel, Switzerland) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and 1 mg/ml Geneticin® (Life Technologies, Carlsbad, USA). Primary fibroblast NHDF-c cells (PromoCell, Heidelberg, Germany) were maintained in DMEM supplemented with 10% heat-inactivated FBS and 2 mM L-glutamine. All cell lines were maintained at 37 °C and 5% CO₂.
HCMV strain AD169 was used for all infections and virus stock was prepared by infecting NHDF-c cells and pelleting the virus from cell supernatant at 100% cytopathic effect. The virus was stored at − 80 °C and titer was measured in plaque assay, performed on NHDF-c cells in carboxymethyl cellulose (CMC) medium.

HT-29 colon carcinoma cells were maintained in DMEM (Lonza) supplemented with 10% heat-inactivated Fetal Calf Serum (FCS)(Serana), 2 mM glutamine (Lonza), 100U penicillin and 100 μg streptomycin (Lonza) at 37 ℃ and 5% CO₂. Cells routinely tested negative for mycoplasma contamination. Cells were seeded at 3 × 10⁵ cells ml^-1 in a 48-well culture plate (VWR) 6 days prior to the assay. Confluent monolayers were washed with DMEM before exposing them to yeast. C. albicans was grown overnight in Sabouraud Dextrose Broth (Sigma)(37 ℃, 200 rpm). 25 × 10⁶ yeast cells ml^-1 were then incubated for 30 min in 0.5 mg mL^-1 calcofluor white stain (Merck, Amsterdam, the Netherlands) (37 ℃, 200 rpm), washed and resuspended at 8 × 10⁶ cells ml^-1 in DMEM without phenol red (Lonza). Next, HT-29 cells were exposed to 4 × 10⁵ yeast cells in 200 μl for 1 h at 37 ℃. Non-adhered cells were washed away and fluorescence intensity was measured on a Clariostar plate-reader (λ_exc = 350/15 nm, λ_em = 433/20 nm). All signals were blank (i.e. only HT-29 cells) corrected, and adhesion is expressed relative to unwashed yeast-exposed cells: (intensity adhered cells/intensity control well)*100%.

All cell lines were obtained from ATCC and were cultured at 37 °C with 5% CO₂ in an incubator with humidified atmosphere. Undifferentiated R1 mouse embryonic stem cells (CVCL_2167) were plated on mitomycin C-treated mouse embryonic fibroblast (CF-1-MEF, CVCL_5251 ) feeders for 2 passages and then plated on tissue culture dishes coated with 0.1% gelatin (Millipore, Milan, Italy), in DMEM (LONZA, Milan, Italy) supplemented with 15% fetal bovine serum (HyClone, Milan, Italy), 1% non-essential amino acids (NEAA) (GIBCO, Monza, Italy), 1% penicillin/streptomycin (P/S) (GIBCO, Monza, Italy), 1% Glutamax (GIBCO, Monza, Italy), 0.1 mM β-mercaptoethanol (Sigma, Milan, Italy) and 1000 U/mL leukemia inhibitory factor LIF (Millipore). ESC cells were used for maximum 10 passages.
HEK-293T (CVCL_0063) cells used for virus production, NIH3T3 (CVLC_0594), C2C12(CVCL_0188), and MEF cells used for ES feeder were cultured in DMEM (LONZA, Milan, Italy), containing 10% FBS (GIBCO, Monza, Italy), 1% P/S (Invitrogen, Monza, Italy); medium was supplemented with 100 uM β-mercaptoethanol (Sigma, Milan, Italy) for MEF cells.