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5 protocols using napyruvate

1

Derivation of Dendritic Cells and Macrophages from Bone Marrow

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DCs were derived from BMDC as previously described with modifications 41. BM cells were cultured at 106 cells per well in 6‐well plates for 6 days in GM‐CSF–containing medium (pyrogen‐free DMEM [Gibco, Thermo‐Fisher, Waltham, MA] with 20 UI/mL GM‐CSF from conditioned supernatants, 10% FCS [Hyclone, GE Healthcare, Logan, UT], 1× nonessential aminoacids [Gibco], 1 mM Na pyruvate [Gibco], 1× GlutaMAX™[Gibco], 50μM β‐ME [Fluka, Sigma‐Aldrich, St. Louis, MO], and kanamycin 0.1 mg/mL [Gibco]). Cultures media was replaced on day 4 with conditioned media without antibiotic.
Macrophages were derived from BMM as previously described with modifications 42. BM cells were cultured at 10–15 × 106 cells in 150 mm Petri dishes for 7 days in M‐CSF–containing medium (pyrogen‐free DMEM with 20% l‐cell conditioned media, 10% FCS, 5% Horse serum [Sigma], 1 mM Na pyruvate, 1.5 mM l‐glutamine [Corning, Corning, NY], 1× penicillin/streptomycin [Gibco]). BMM were harvested on day 7 and replated at 106 cells per well in 6‐well plates in conditioned media without antibiotic.
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2

Culturing Ocular Melanoma Cell Lines

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MP41 and MP46 were purchased from American Type Culture Collection (ATCC). 92.1 cells were gifted by Dr. Martine Jager (Leiden University, Netherlands) [33 (link)]. MEL270 and OMM2.5 were gifted by Dr. Vanessa Morales (University of Tennessee). OCM1 was received from the Institute of Ophthalmology and Visual Sciences (University of Valladolid). Cells were cultured in RPMI-1640 (Corning) supplemented with 10% Fetal Bovine Serum, 10 mM HEPES, 2 mM Corning glutaGRO, 1 mM NaPyruvate, 0.1% 10 U/mL penicillin and 10 μg/mL streptomycin (all from Corning), and 10 μg/mL insulin (Roche) in a 37 °C and 5% CO2 atmosphere.
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3

Salmonella Infection in Macrophage Model

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THP-1 cells were obtained from ATCC (American Type Culture Collection: TIB-202) and grown in suspension in RPMI +Glutamax supplemented with 10% (v/v) FBS in a humidified 37°C, 5% CO2 incubator. All cell culture reagents were from Life Technologies, unless otherwise stated. Low passage (passage 15 or less) cells were plated in either 8-chamber Ibidi dishes (Ibidi) or 24-well tissue culture treated plates (Costar) in the presence of phorbol 12-myristiate-12 acetate (PMA, Sigma-Aldrich). For cells plated 5 days prior to infection, PMA was removed after 2 days of treatment. Primary human monocytes were isolated using negative selection (Dynabeads Untouched Human Monocyte Isolation Kit, Life Technologies) from apharesed whole blood from healthy donors and plated in 8-well Ibidi dishes 7 days prior to infection in RPMI supplemented with 5% (v/v) Male AB human serum (Sigma), 2 mM L-glutamine (Cellgro), 1 mM NaPyruvate (Cellgro), 1X MEM NEAA, 1 mM Hepes and 100 ng/mL macrophage colony-stimulating factor (MCSF) (Peprotech). Media was refreshed on day 3 and day 5. Salmonella enterica serovar Typhimurium SL1344 wild type [29 (link)] and constitutively expressing mCherry [30 (link),31 (link)] have been previously described.
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Rat Alveolar Cell Culture Assay

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Materials were obtained from the following suppliers: female rat non-cancerous type-II alveolar cells (CC-149, L2 cells) and fetal bovine serum (FBS), ATCC; low glucose DMEM, Hyclone. Phenol red-free low glucose DMEM, 17β-estradiol (E2) and 3,3′, 5-triiodo-L-thyronine sodium salt (T3), Sigma-Aldrich; charcoal-stripped FBS and 100X antibiotic-antimycotic, Invitrogen; Trypsin-EDTA .05%, VWR; Na-pyruvate, Cellgro; Hanks buffered saline solution (HBSS), Lonza; MTT, PGE2, and Apotox-Glo Triplex assay kits from Roche, Cayman Chemical, and Promega, respectively. Promega generously provided GSH/GSSG-Glo assay prior to public release.
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5

3D Microfluidic Spheroid Culture of MPM Cells

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MPM tumor specimens, MSTO-211H or LP9 cells were used to prepare MPM spheroids analogous to previous preparations [40 (link)] In brief, fresh MPM tumor specimens were minced in Dulbecco’s Modified Eagle’s medium (DMEM) (containing 10% FBS, 100 mmol/L Na pyruvate, Corning CellGro, Tewksbury, MA, USA), 100 U/mL collagenase type IV (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), and 15 mmol/L HEPES (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA). Visible red blood cells were removed using red blood cell lysis buffer (Boston Bio-Products, Ashland, MA, USA) and strained over 40 μm filters. Cells were maintained in ultralow-attachment tissue culture plates before injection into the culture chamber. Cell preparations or cell lines were pelleted and resuspended in type I rat tail collagen (2.5 mg/mL, Corning, Tewksbury, MA, USA) following the addition of 10× PBS with phenol red (pH 7.0–7.5). The spheroid–collagen mixture was then injected into the center gel region of the 3D microfluidic culture device. Collagen hydrogels were hydrated with media with or without indicated drugs or control treatments after 30 min at 37 °C. Microfluidic culture as well as live/dead staining and quantification of cells was performed as previously described [31 (link),40 (link)].
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