Penicillin and streptomycin mixture

Peripheral blood mononuclear cells (PBMC) were isolated from buffy coat by Ficoll gradient centrifugation (GE healthcare) and washed three times in PBS. T cells were negatively selected with magnetic-assisted cell sorting (MACS) from PBMC with the use of CD8⁺ T cell isolation kit or Pan T cell isolation kit (Miltenyi Biotec). Primary human T cells were cultured in RPMI 1640 medium (Gibco), supplemented with 10% heat inactivated FBS (Gibco), and 1% Penicillin and Streptomycin (Gibco). Monocytes were positively selected with MACS by CD14 Microbeads (Miltenyi Biotec) and were cultured in RPMI 1640 medium (Gibco), supplemented with 10% heat inactivated FBS (Gibco), and 1% Penicillin and Streptomycin mixture (Gibco).

SK-MEL-28-N1 (referred as to N1 hereafter), a GD3-negative cell line isolated from SK-MEL-28 human melanoma cells, was established previously53 (link), and was used as recipient cells for the glycan remodelling. The major ganglioside in N1 cells is GM3. N1 cells stably expressing GD3 (G5 and G11) and mock transfectants (V4 and V9) were generated previously in our lab25 (link). GM2-expressing and GD2-expressing cell lines were established by transfecting N1 cells and GD3-expressing cells with human B4GALNT1 cDNA expression vectors54 (link), respectively. DNA transfection was conducted by using Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol. Afterwards, GM2- and GD2-expressing cells were sorted by flow cytometry using anti-GM2 mAb and anti-GD2 mAb, respectively, and clones were then obtained by limiting dilution. GM1-expressing cell lines were established by trasfecting GM2-expressing cells with B3GALT4 cDNA51 (link) as above, followed by flow cytometric isolation of cells that bind biotin-CTxB. These melanoma cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 7.5% FBS, 1% penicillin and streptomycin mixture (Gibco) and 400 μg/ml of G418 at 37 °C in an incubator with 5% CO₂. For experiments, cells were seeded into 6-well plates at a density of 2 × 10⁵ cells per well or 100-mm dishes at a density of 1 × 10⁶ cells per dish.

All cell lines were purchased from the National Collection of Authenticated Cell Cultures (China). 786-O and Caki-1 cells were cultured in RPMI 1640 (HyClone), ACHN cells were cultured in MEM (HyClone), and 293T cells were cultured in DMEM (Gibco) media. All cell culture media were supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin and streptomycin mixture (Gibco). Cell cultures were maintained in a humidified incubator at 37 °C and 5% CO₂.

Human small intestinal epithelial cell line (FHs74Int), (American Type Culture Collection (ATCC), Manassas, VA, USA) was cultured in Hybri-Care Medium (ATCC) supplemented with 30 ng/mL EGF, 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) and 1% penicillin and streptomycin mixture (Life Technologies) under standard cell culture conditions (37 °C, humidified, 5% CO₂/95% air environment).
For real time RT-PCRs the cells were seeded into 6 or 96 well plates at a density of 10⁵ or 10⁴ cells/well and treated with IL-1β (100 ng/mL), TNF-α (10 ng/mL), TGF-β (0.5 nM), or IL-17 (100 ng/mL) on 6 well plates (n = 6 well/treatment group), or with IL-24 (0.1 ng/mL) and H₂O₂ (1000 µM) on 96 well plates (n = 5 well/treatment group) for 24 h, respectively.
For MTT and LDH assays the cells were seeded into 96-well plates at a density of 10⁴ cells/well (n = 5 well/treatment group) and treated with IL-24 (0.1 ng/mL) and H₂O₂ (200, 400, 600, 800 or 1000 µM) (Sigma-Aldrich) for 24 h.
The cells were seeded into 6 well plates at a density of 3 × 10⁵ cells/well (n = 3 well/treatment group) for the Annexin V apoptosis assay, and treated with IL-24 (0.1 ng/mL) and H₂O₂ (1000 µM) for 48 h.
Vehicle treated cells served as controls in all experiments.

Primary hASCs were obtained from ScienCell Company (San Diego, CA, USA). All cell-based in vitro studies were repeated three times using hASCs from three healthy donors. DMEM, FBS, and 100× penicillin and streptomycin mixture were obtained from Gibco (Grand Island, NY, USA). Human ASCs were cultured with 5% CO₂ atmosphere at 37 °C in proliferation medium (PM), which consisted DMEM, 10% (v/v) FBS and penicillin/streptomycin. The OM comprised DMEM containing 10% (v/v) FBS, penicillin/streptomycin, 10 nM dexamethasone, 10 mM β-glycerophosphate, and 0.2 mM L-ascorbic acid. The AM was comprised of DMEM containing 10% (v/v) FBS, penicillin/streptomycin, 10 μM insulin, 100 nM dexamethasone, 200 μM indomecin, and 500 μM 3-isobutyl-1-methylxanthine (IBMX).

Primary human BMMSCs and human adipose-derived mesenchymal stem cells (hASCs) were purchased from ScienCell Company (San Diego, CA, USA). All cell-based in vitro studies were repeated three times using MSCs from three donors. All materials were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. This study was approved by the Institutional Animal Care and Use Committee of the Peking University Health Science Center (LA2014233), and all experiments were performed in accordance with the approved guidelines.
FBS, MEM, DMEM, and 100× penicillin and streptomycin mixture were purchased from Gibco (Grand Island, NY, USA). Human BMMSCs and ASCs were cultured in proliferation medium (PM), consisting of 10% (v/v) FBS, penicillin/streptomycin, and fresh MEM (for hBMMSCs) or DMEM (for hASCs), with 5% CO₂ atmosphere at 37 °C. The OM comprised fresh DMEM or MEM containing 10 nM dexamethasone, 10 mM β-glycerophosphate, 0.2 mM l-ascorbic acid, 10% (v/v) FBS, and penicillin/streptomycin. TNF-α was purchased from R&D Systems (Minneapolis, MN, USA), and BAY117082 was purchased from Selleck (Houston, TX, USA). Cells at the fourth to sixth passage were used for the in vitro experiments.