For western blotting, HEK293T cells were grown in DMEM medium (Sigma-Aldrich, D0819) supplemented with 10% FCS (Sigma-Aldrich, F0392), 1% sodium pyruvate (Sigma-Aldrich, S8636) and 1% penicillin/streptavidin (Sigma-Aldrich, P4333). For immunofluorescence analysis, hTERT RPE-1 and IMCD-3 cells were grown in DMEM/F12, supplemented with 10% FCS (Sigma-Aldrich, F0392), 1% sodium pyruvate (Sigma-Aldrich, S8636) and 1% penicillin/streptavidin (Sigma Aldrich, P4333). IMCD-3 cells stably expressing WASF3 were further supplemented with 400 g/μl hygromycin (Sigma-Aldrich, H0654). All cells were cultured at 37°C and under 5% CO2.
F0392
Manufactured by Merck Group
Sourced in Germany
F0392 is a laboratory instrument designed for centrifugation. It is capable of separating different components of a liquid sample by applying centrifugal force.
Automatically generated - may contain errors
Lab products found in correlation
F2442, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Molt 4, by American Type Culture Collection (2 mentions)
Jurkat, by American Type Culture Collection (2 mentions)
Molt 3, by American Type Culture Collection (2 mentions)
Kopt k1, by American Type Culture Collection (2 mentions)
Anti shmt1 12612, by Cell Signaling Technology (2 mentions)
Shmt2 12762, by Cell Signaling Technology (2 mentions)
Anti β actin a3854, by Merck Group (2 mentions)
Hct116, by American Type Culture Collection (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Na934, by Merck Group (2 mentions)
Hpb all, by Leibniz Institute DSMZ (2 mentions)
Dnd41, by Leibniz Institute DSMZ (2 mentions)
Stericup sterile vacuum filter, by Merck Group (2 mentions)
S11150, by Atlanta Biologicals (2 mentions)
Glutamine, by Merck Group (2 mentions)
G7513, by Merck Group (2 mentions)
F9665, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
25 protocols using f0392
1
Western Blotting and Immunofluorescence of Cell Lines
2
Genetically Modified Mouse Fibroblasts
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PKM2 knockout mouse embryonic fibroblasts MEFs and their matched wild-type counterparts were obtained from the laboratory of Dr. Matthew Vander Heiden (Massachusetts Institute of Technology, Cambridge, MA34 (link). Standard cancer cell lines (A375, SK-MEL-28, LNCaP, CAL-51, A549, B16, and HeLa), Aprt−/− Hprt−/− A9 mouse fibroblasts (CCL-1.4) and their parental cell line (CCL-1) were from ATCC. All cell lines were maintained in DMEM (Corning/Cellgro, 10-017-CV) containing 10% fetal bovine serum (FBS) at 37 °C and 5% CO2. Treatments with purine and pyrimidine inhibitors were performed in the presence of 10% dialyzed FBS (Sigma, F0392) to avoid the presence of nucleosides and nucleobases. Cells were treated with MTX, LTX, leflunomide, 5-fluorouracil (5-FU), 6-MP, mizoribine, TEPP-46, BI-4984, NCT-503, and SHIN1 as indicated in the figure legends. Exogenous adenine and inosine were added at 50 µM concentrations for 1 h unless otherwise indicated.
3
Cell Culture Media Optimization
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All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM/HIGH GLUCOSE, SH30022.01, HyClone, GE Healthcare, Logan, UT, USA) and Ham’s nutrient mixture F1 medium (SH30026FS, HyClone, GE Healthcare) 1:1 supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 20 mM HEPES, and 10% fetal bovine serum (FBS, SV30160.03 HyClone, GE Healthcare) as previously described [11 (link)]. To prepare media with lower amino acid content, normal DMEM/Ham’s F12 medium was diluted with the corresponding volume of amino acid-free DMEM. Amino acid-deprived media were supplemented with 10% dialyzed FBS (10,000 Mwt cut-off; F0392, Sigma-Aldrich).
4
Cultivation of Human Glioblastoma Cell Lines
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Human glioblastoma U251MG and U87MG cell lines were obtained from the Cell Lines Service (Eppelheim, Germany). Cells were cultivated at 37 °C with 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM; Gibco 31966021, Waltham, MA, USA) supplemented with GlutaMAX-1, 10% heat inactivated fetal bovine serum (FBS; Gibco 10500064) and antibiotics, 1% penicillin/streptomycin (Gibco 15140122). For all experiments, we formulated DMEM (Sigma-Aldrich D9443) with or without 0.4 mmol/L arginine (complete medium (CM) and arginine-free medium (AFM), respectively), supplemented with 5% dialyzed serum (Sigma-Aldrich F0392).
5
Establishment and Characterization of Human Cancer Cell Lines
6
Quantification of Newly Synthesized Proteins
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Human U2OS NMD reporter cells were treated with DMSO or ouabain in methionine-free media (CellGro, 17-204-CI) supplemented with 2 mM L-glutamine, 10% dialyzed FBS (Sigma, F0392), and 35S-labeled methionine (10 µCi ml−1) for 24 h prior to protein collection. Protein samples from the same number of cells were run on two separate SDS-PAGE gels, with one for 35S autoradiography analysis (newly synthesized proteins) and one for Coomassie blue staining (total protein levels). Images were quantified using ImageJ and autoradiography intensity was normalized to the total protein level (Coomassie blue intensity).
7
Culturing Breast and Prostate Cancer Cells
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MCF7 breast cancer cells and PC3 prostate cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO-11995) supplemented with 10% fetal bovine serum and 1 × antibiotics (penicillin, 10,000 UI/ml and streptomycin, 10,000 UI/ml). To prepare amino acid deficient media, Earle’s balanced salt solution was added with 4.5 g/L glucose (Sigma-Aldrich), MEM vitamin solution (Invitrogen), 0.37mM sodium bicarbonate (Sigma-Aldrich), 24.8 μM ferric nitrates (Sigma-Aldrich), with deficiency of one (or all) amino acid, and supplemented with 10% dialyzed FBS (10,000 MW cutoff, Sigma (F0392)) and 1X antibiotics. The cells were maintained in a humidified incubator at 37°C and 5% CO2. Additional materials and methods were listed in S1 Text .
8
SILAC-Based Quantitative Proteomics
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For quantitative mass spectrometry (MS) analysis, SILAC cells were labeled by culturing them in DMEM without Arg/Lys (A14431-01), 200 µM L-glutamine, 100 U/ml penicillin streptomycin, 110 mg/l (1 mM) sodium pyruvate, and 10% 10 kD dialyzed serum (F0392; Sigma-Aldrich). The media for light labeling was supplemented with Arg 0 (84 mg/ml, A6969; Sigma-Aldrich) and Lys 0 (146 mg/ml, L8662; Sigma-Aldrich), and the media for heavy labeling was supplemented with Arg10 (CNLM-539-H; Cambridge Isotope Laboratories) and Lys8 (CNLM-291-H; Cambridge Isotope Laboratories). Incorporation of the isotopes was confirmed after six passages. siControl and siMASTL#6 silenced samples were prepared two times with light- and heavy-labeled cells, obtaining four experimental replicates. For each independent experiment, siControl and siMASTL-silenced samples were mixed using a label-swap replication strategy (i.e., siControl-light mixed with siMASTL silenced-heavy generates forward replicate 1; siMASTL silenced-light with siControl-heavy generates reverse replicate 1, etc.). 48 h after silencing the cells were lysed with 4% SDS, 100 mM DTT, and 100 mM Tris-HCl, pH 7.6, and boiled for 5 min at 95°C. Samples were sonicated, centrifuged at 13,000 g for 10 min at 4°C, and transferred into a clean tube.
9
Establishment and Characterization of Human Cancer Cell Lines
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Human colon cancer cell line HCT116 and human T-ALL cell lines Molt4, Molt3, Jurkat and KOPT-K1 were obtained from ATCC. HPBALL and DND41 human T-ALL cell lines were obtained from DSMZ (The Leibniz Institute). The CUTLL1 NOTCH1-dependent T-cell lymphoblastic cell line has been previously described (ref. 24 (link)). SHMT1 and SHMT2 were knocked out in HCT116 lines using CRISPR/Cas9 nickase method as previously described (ref. 4 (link),18 (link)). Adherent cell lines were subcultured in 5% CO2 at 37 °C using DMEM (CellGro 10–017; Mediatech) supplemented with 10% FBS (F2442; Sigma-Aldrich); suspension cell lines were subcultured in 5% CO2 at 37 °C in RPMI-1640 (11875; Gibco) with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. For all experiments, media supplemented with 10% dialyzed FBS was used (F0392; Sigma-Aldrich). Cell lines were regularly tested for mycoplasma. Antibodies were used according to their manufacturers’ directions. Anti-SHMT1 (12612) and SHMT2 (12762) were obtained from Cell Signaling Technologies (1:1,000 dilution). Anti-β-ACTIN (A3854) was obtained from Sigma-Aldrich (1:50,000 dilution). Secondary antibody coupled to horseradish peroxidase (NA934) was obtained from Sigma-Aldrich. Signal was detected using enhanced chemiluminescence (34578, Thermo Scientific).
10
Folate Metabolism in Cell Culture
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