Ficoll-Paque Plus (VWR International, Radnor, PA, USA) was used to isolate peripheral blood mononuclear cells (PBMCs) from collected blood as previously described [20] (link). The generation of LCLs was carried out as previously described [9] (link). Briefly, PBMCs were incubated for 1 hr at 37°C with supernatant from the EBV B95.8 cell line, after which the cells were suspended in RPMI-1640 (Lonza, Basel, Switzerland) containing 10% fetal bovine serum (FBS, Sigma Aldrich, St Louis, MO, USA), 2 mM L-glutamine (Life Technologies), and 2 μg per ml cyclosporin A (Sigma Aldrich). The cells were plated at densities of 2.5 × 106 or 5 × 106 cells per well in 48-well plates. The media was supplemented weekly until the cells were expanded into a 25 cm2 flask. LCLs were used then cryopreserved in 10% DMSO (MP Biomedical, Irvine, CA, USA) 50% FBS and RPMI-1640. The LCLs undergo routine mycoplasma testing.
Rpmi 1640
Manufactured by MP Biomedicals
Sourced in United States
RPMI 1640 is a cell culture medium commonly used for the in vitro cultivation of various cell types, including human and animal cells. It is a complex and balanced formulation that provides the necessary nutrients and growth factors required for cell proliferation and maintenance. The medium is designed to support the optimal growth and survival of a wide range of cell lines.
Automatically generated - may contain errors
Lab products found in correlation
35s methionine cysteine, by PerkinElmer (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cyclosporin a, by Merck Group (1 mentions)
Ficoll paque plus, by Avantor (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
Dimethyl sulfoxide (dmso), by MP Biomedicals (1 mentions)
Protein g sepharose bead, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Lymphocyte separation medium, by Corning (1 mentions)
Recombinant human il 2, by Merck Group (1 mentions)
Pha p, by Merck Group (1 mentions)
3h thymidine, by MP Biomedicals (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Protease inhibitor mixture, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by MP Biomedicals (1 mentions)
E g7 ova, by American Type Culture Collection (1 mentions)
8 protocols using rpmi 1640
1
Establishing EBV-Transformed Lymphoblastoid Cell Lines
2
Radioactive Protein Labeling and Immunoprecipitation
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Freshly cultured parasites were washed and resuspended in methionine-free RPMI 1640 (MP Biomedicals Inc., Aurora, OH). 200 µCi/mL of [35S] methionine/cysteine (PerkinElmer, Boston, MA) was added and parasites were incubated at 37°C for 2 h. Parasites were lysed in NETT buffer [10 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.1% Triton X-100] using a protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA)) and centrifuged to collect the supernatant. Lysates were pre-cleared with Protein G-Sepharose beads (GE Healthcare, Waukesha, WI) before antibody addition. Protein G-Sepharose beads were added and washed extensively with NETTS (10 mM Tris, pH 7.5, 500 mM, NaCl, 5 mM EDTA and 0.1% Triton X-100) and NETT buffers. Protein was eluted from the beads using elution buffer [10 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA], boiled and run on SDS-PAGE gel. Gels were fixed with fixing solution [25% Isopropanol alcohol; 10% Acetic Acid] for 30 min and enhanced with Amplify™ fluorographic solution (GE Healthcare, Waukesha, WI) for 45 min, dried under vacuum, and exposed to X-ray film for autoradiography.
3
Aspergillus fumigatus Spore Isolation
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A. fumigatus (ATCC 204304) was grown on YPD agar slants in an incubator at 35°C until the formation of conidia (approximately 48 hours). Conidia were harvested by overlaying the slant with sterile media (RPMI 1640, with L-glutamine, without sodium bicarbonate, MP Biomedicals, Solon, OH, USA) containing 0.01% TWEEN 20 (Sigma-Aldrich, St. Louis, MO, USA). A conidium suspension was prepared by gently probing the colonies with the tip of a transfer pipette. After allowing large particles to settle for 3 to 5 minutes, the suspension was washed twice with media.
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A. fumigatus (ATCC 204304) was grown on YPD agar slants in an incubator at 35°C until the formation of conidia (approximately 48 hours). Conidia were harvested by overlaying the slant with sterile media (RPMI 1640, with L-glutamine, without sodium bicarbonate, MP Biomedicals, Solon, OH, USA) containing 0.01% TWEEN 20 (Sigma-Aldrich, St. Louis, MO, USA). A conidium suspension was prepared by gently probing the colonies with the tip of a transfer pipette. After allowing large particles to settle for 3 to 5 minutes, the suspension was washed twice with media.
4
Isolation and Transduction of Mouse Islets
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An abdominal incision was performed to 8-week old male C57BL/6 mice under anesthesia to access the pancreas, which was inflated and harvested. Digestion was carried out with collagenase (CIzyme RI from Vitacyte, Indianapolis, IN). Gradient centrifugation and gravity sedimentation of islets were carried out in lymphocyte separation medium (Corning) and Hank’s balanced salt solution (ThermoFisher). Islets were handpicked under a microscope, counted and cultured in RPMI 1640 (MP Biomedicals, Santa Ana, CA) supplied with 10% FBS (ThermoFisher) in Petri dishes at 37 °C and 5% CO2. Intact islets underwent two rounds of adenoviral transduction (MOI as stated, ~1,500 cells/islet) 48 h apart.
5
Assessing IL-12 Bioactivity via T-Cell Proliferation
6
Exposure of Polarized Macrophages to CeO2 NPs
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Three experiments were conducted with polarized human macrophages exposed to 10 μg/mL (as cerium) CeO2 NPs for 4.5 h. Nine experiments were conducted with polarized RAW cells exposed to 0 (PBS), 1, 3, or 10 μg/mL (as cerium) CeO2 NPs or 10 μg/mL Ce-ion for 6 or 24 h. To expose the cells, the medium was replaced with a phosphate-free medium (DMEM (or MP Biomedicals RPMI 1640 without phosphate and with 0.85 G/L sodium bicarbonate)) with 10% FBS. Complete DMEM medium has 0.915 mM phosphate. Nine percent FBS has ~8 µg/mL phosphorus (~0.25 mM phosphate). Total phosphate in DMEM + FBS was ~1.16 mM; in phosphate-free medium, ~0.25 mM. The CeO2 NPs and Ce-ion (Aldrich, Burlington, MA, USA, ICP/DCP standard solution) were introduced in 110 mM citric acid at pH 7.4. Cells were harvested after 6 or 24 h exposure, or the medium replaced with DMEM with 10% FBS and harvested up to 2 weeks later. For microscopy, they were washed with PBS and preserved in 2.5% PBS-buffered glutaraldehyde. Cells were processed for light microscopy and EM visualization and were not stained nor were heavy metals (e.g., osmium) added.
7
Metabolic Labeling of Parasitic Proteins
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Freshly cultured parasites were washed and re-suspended in methionine-free RPMI 1640 (MP Biomedicals Inc., Aurora, OH). 200 µCi/mL of [35S] methionine/cysteine (PerkinElmer, Boston, MA) was added, and parasites were incubated at 37°C overnight. The supernatant was collected by centrifugation at 14,000 rpm for 15 min and a protease inhibitor mixture (Sigma-Aldrich, St Louis, MO) was added. The parasite supernatant was pre-cleared with protein G (GE Healthcare, Waukesha, WI) before to use.
8
Murine Dendritic Cell and T Lymphoma Protocols
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DC2.4 cell, which is an immature murine DC line, was provided from Dr. K. L.
Rock (Harvard Medical School, USA) and were grown in RPMI-1640 supplemented with 10% FBS (MP Biomedical, Inc.), 2 mM L-glutamine, 100 mM nonessential amino acid, 50 M 2-mercaptoethanol (2-ME, Gibco) and antibiotics at 37 °C [35] .
E.G7-OVA, which is a chicken egg OVA gene-transfected clone of C57BL/6 mice-derived T lymphoma and which presents OVA with MHC class I molecules, was obtained from the American Type Culture Collection (Manassas, VA) [36] .
Rock (Harvard Medical School, USA) and were grown in RPMI-1640 supplemented with 10% FBS (MP Biomedical, Inc.), 2 mM L-glutamine, 100 mM nonessential amino acid, 50 M 2-mercaptoethanol (2-ME, Gibco) and antibiotics at 37 °C [35] .
E.G7-OVA, which is a chicken egg OVA gene-transfected clone of C57BL/6 mice-derived T lymphoma and which presents OVA with MHC class I molecules, was obtained from the American Type Culture Collection (Manassas, VA) [36] .
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