Leishmania donovani strain AG83 (MHOM/IN/1983/AG83), used in this study was maintained routinely in golden hamsters by repeated passage to sustain its virulence. Amastigotes were isolated from the spleen of infected hamster and were made to undergo transformation from amastigotes to promastigotes routinely, prior to infection as described earlier (41 (link)). Leishmania parasites were grown at 22°C in normal RPMI media (Invitrogen, USA) and supplemented with 10% heat-inactivated dialyzed FBS (Invitrogen, USA), 25 mM HEPES, pH 7.4, 4 mM NaHCO3, 100 U/ml of penicillin G-sodium (Sigma-Aldrich), and 100 mg/ml of streptomycin (Sigma-Aldrich), and these parasites were used for our experimental purpose. Levels of LPS in parasite stock preparations were found to be below the limit of detection [<0.1 endotoxin U/ml (LAL-test; BioWhittaker, Inc., Walkersville, MD, USA)].
Dialyzed
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dialyzed is a lab equipment product designed to facilitate the removal of small molecules from a sample. It operates by allowing the selective diffusion of molecules through a semi-permeable membrane, while retaining larger molecules of interest. This process is commonly used in biochemistry, molecular biology, and biotechnology applications to purify and concentrate macromolecules such as proteins, nucleic acids, or carbohydrates.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Co2 incubator, by Thermo Fisher Scientific (3 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Hepes, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Rpmi media, by Thermo Fisher Scientific (1 mentions)
Penicillin g sodium, by Merck Group (1 mentions)
Lal test, by Lonza (1 mentions)
Streptomycin, by Merck Group (1 mentions)
L lysine, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Gammacell 3000 elan, by Nordion (1 mentions)
Glucose colorimetric detection kit, by Thermo Fisher Scientific (1 mentions)
Lactate assay kit, by Merck Group (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
D glucose, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Dmem medium, by Corning (1 mentions)
Silac dmem flex media, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
15 protocols using dialyzed
1
Culturing Leishmania donovani Parasites
2
Versatile Cell Culture Protocols for Diverse Cell Lines
3
Radiation Response in MDA-MB-231 Cells
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MDA-MB-231 cells were maintained in l -lysine-depleted RPMI 1640 (Invitrogen, Grand Island, NY, USA) supplemented with 10% dialyzed FBS (Invitrogen, Carlsbad, CA, USA) and 0.1 mg/mL heavy [U-13C6] or light l -lysine (Invitrogen). Every 3–4 days, cells were split and media replaced with the corresponding light or heavy labeling medium. After approximately six doubling times, cells achieved almost 100% incorporation of amino acid isotopes. Cells grown with light l -lysine (1 × 106 cells/100 mm dish) were exposed to 10 Gray (Gy) 137Cs γ-radiation (Gammacell 3000 Elan, MDS Nordion, Canada) and harvested after 48 h.
4
Versatile Cell Culture Protocols for Diverse Cell Lines
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HEK293A, HEK293P, HEK293T, H2.35, NIH3T3, C2C12, and HeLa cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) (Invitrogen) and 50 µg/ml penicillin/streptomycin in a humidified incubator with 5% CO2. MCF10A cells were cultured in DMEM/F12 supplemented with 5% horse serum, 20 ng/ml EGF, 0.5 µg/ml hydrocortisone, 10 µg/ml insulin, 100 ng/ml cholera toxin, and 50 µg/ml penicillin/streptomycin. HepG2 cells were cultured in RPMI1640 supplemented with 10% FBS and 50 µg/ml penicillin/streptomycin. For glucose starvation, cells were washed twice with PBS and then incubated in DMEM without glucose and pyruvate (0 g/L glucose, 0 mM pyruvate; Invitrogen) supplemented with 10% FBS, Dialyzed (Invitrogen). Lats1 KO MEF cells, provided by Dr. Dae-SiK Lim (Korea Advanced Institute of Science and Technology), were maintained in DMEM with 10% FBS 39 (link). AMPKα WT and AMPKα DKO MEFs, a gift from Dr. Benoit Viollet, were cultured in DMEM medium with 10% FBS 40 (link). PolyJet™ DNA In Vitro Tranfection reagent (SignaGen Lab.) was used for transfection. siRNAs were synthesized by Dharmacon and delivered into cells using RNAiMAX (Invitrogen) according to manufacturer’s instructions.
5
Glucose Metabolism in CLL Cells
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Isolated CLL cells were plated at density of 107 per mL in Glucose-free RPMI1640 (Gibco) supplemented with 10% [vol/vol] dialyzed FCS (Gibco), 2.5% [vol/vol] 1M HEPES (Sigma), 1% [vol/vol] Glutamax (Gibco), and 0.1% [vol/vol] 0.05M β-mercaptoethanol (Sigma) with the addition of 4 mmol/L D-glucose (Sigma). Spent media was collected after 2 days, and the residual glucose and lactate concentrations were measured using a glucose colorimetric detection kit (Invitrogen) and lactate assay kit (Sigma) according to the manufacturer’s instructions.
6
Nutrient Starvation Assay Protocol
7
SILAC Time-Course Proteome Profiling
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HeLa Kyoto cells (from S. Narumiya, RRID: CVCL_1922, see also Supplementary Data 5 ) were cultured according to standard tissue culture techniques in SILAC DMEM Flex Media (Gibco) containing either “Light” (12C and 14N-labeled, Arg0 and Lys0, Thermo) or “Heavy” (13C and 15N-labeled, Arg10 and Lys8, Silantes) labeled arginine and lysine, 1 mg ml−1 glucose, 10% dialyzed FBS (Gibco), and 1 mM L-glutamine. Cells were grown for a minimum of 10 doublings in the respective media before starting a time course to ensure complete labeling. For the SILAC time course 2.3E6 cells were seeded onto 15 cm dishes in Light (replicates 1 through 3) or Heavy medium (replicate 4) and cultured for 24 h to 48 h before medium exchange. A single 15 cm dish was used per time point per replicate. Medium was aspirated and cells washed twice with warm PBS (with Ca and Mg), then medium replaced with Heavy (replicates 1 through 3) or Light medium (replicate 4). For each replicate all dishes were seeded and lysed simultaneously with staggered medium exchange.
Both cell lines used were verified to be mycoplasma free.
Both cell lines used were verified to be mycoplasma free.
8
Culturing Cell Lines and Toxoplasma gondii
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The cell lines (Ana‐1 and Vero) and T. gondii RH strain were kept in the Laboratory of Veterinary Molecular and Immunological Parasitology, Nanjing Agricultural University, China.
Murine macrophages (Ana‐1) utilized in cellular function evaluations and Vero cells utilized to sustain T. gondii were cultured in Dulbecco's modified Eagle's medium (Gibco, New York City, NY) augmented with 10% dialyzed fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco) in a CO2 incubator (Thermo, Waltham, MA) at 37 °C.
Murine macrophages (Ana‐1) utilized in cellular function evaluations and Vero cells utilized to sustain T. gondii were cultured in Dulbecco's modified Eagle's medium (Gibco, New York City, NY) augmented with 10% dialyzed fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco) in a CO2 incubator (Thermo, Waltham, MA) at 37 °C.
9
Starvation Conditions for Cell Culturing
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Cells were plated at 21,000 cells per cm2 in 6-well plates in normal growth media. After 24 h cells were washed twice with phosphate buffered saline (PBS) and treated with the respective starvation or non-starvation media. For the initial screening, DMEM or SILAC RPMI 1640 medium (Gibco) lacking glucose and glutamine were supplemented with 10 mM or 0.2 mM glucose (Sigma-Aldrich, St. Louis, MO, USA), 2 mM glutamine and 0% or 10% dialyzed FBS (Gibco). SILAC RPMI was additionally supplemented with arginine and lysine (Sigma-Aldrich) at concentrations present in normal RPMI 1640. For serine/glycine and glucose starvation, DMEM without glucose, glutamine, serine and glycine, containing standard cystine (Biomol, Hamburg, Germany) was supplemented with serine, glycine and glucose (Sigma-Aldrich) at the indicated concentrations. glutamine was added at a concentration of 2 mM. In some experiments, custom-made DMEM medium lacking glucose, glutamine, serine, glycine, cystine, cysteine, pyruvate and phenol red (Cell Culture Technologies, Gravesano, Switzerland) was used. For the supplementation with different levels of cystine, a 100 mM stock in 1 M HCl was freshly prepared. During the course of experiments media were refreshed every 24 h, unless indicated otherwise.
10
HEK293T Cell Starvation, Stimulation, and Transfection
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Cell culture, amino acid starvation, stimulation, and transient transfection HEK293T cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Wisent) supplemented with 10% fetal bovine serum (FBS) (Wisent) and 1% streptomycin-penicillin (Wisent) in a humidified atmosphere containing 5% CO 2 at 37 C. For amino acid starvation, cells seeded 24 h before starvation were washed by pre-warmed PBS and incubated with amino acid-, leucine-, and threonine-free DMEM (manufactured by Wisent) supplemented with 10% dialyzed FBS (GIBCO). For amino acid stimulation, a concentrated solution, which is prepared from individual amino acid powder, was added to the culture medium at the same concentration as in the complete DMEM (#319-005-CL) (Wisent). Transient transfection of plasmids was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
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