To establish hyperglycemic conditions, D‐(+)‐glucose (D‐glucose, G7021, Sigma, United States) was added directly to EC media (contains 5.5 mm D‐glucose) to obtain the desired glucose concentration (5.5–25.5 mm D‐glucose). L‐(−)‐glucose (L‐glucose, L5500, Sigma, United States) was used as an osmotic control for D‐glucose. Like the D‐glucose treatment conditions, 10 mm of L‐glucose was added to EC media. For ReN cells, SILAC Advanced DMEM/F12 Flex media (no glucose, A2494301, Gibco, United States) supplemented with 1000 mg L−1 (5.5 mm ) D‐glucose (G7021, Sigma, United States), 365 mg L−1 L‐Glutamine (25030149, Gibco, United States), 91.25 mg L−1 L‐lysine (L8662, Sigma, United States), and 147 mg L−1 L‐arginine (A6969, Sigma, United States) was added because DMEM/F12 (11320‐033, Gibco, United States) had high concentration of glucose (3151 mg L−1, 17.5 mm ).
L8662
Manufactured by Merck Group
Sourced in United States
The L8662 is a laboratory device designed for general scientific applications. It is a versatile piece of equipment that can be used for a variety of tasks in research and development settings. The core function of the L8662 is to perform specific laboratory procedures and analyses, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
A6969, by Merck Group (3 mentions)
L8912, by Merck Group (3 mentions)
Cnlm 291 h, by Cambridge Isotopes (2 mentions)
Cnlm 539 h, by Cambridge Isotopes (2 mentions)
C7629, by Merck Group (2 mentions)
A8094, by Merck Group (2 mentions)
G7021, by Merck Group (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Canto 2, by BD (1 mentions)
F0392, by Merck Group (1 mentions)
G8796, by Merck Group (1 mentions)
Silac dmem, by Merck Group (1 mentions)
A5131, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
L leu, by Merck Group (1 mentions)
L met, by Merck Group (1 mentions)
M5308, by Merck Group (1 mentions)
7 protocols using l8662
1
Hyperglycemic Conditions in Cell Culture
2
Kynurenine Uptake Assay in Cells
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HBSS was pre-warmed to 37 °C and kynurenine (final concentration 200 μM, K8525, Sigma), BCH (final 10 mM, A7902, Sigma), leucine (final 5 mM, L8912, Sigma) and lysine (final 5 mM, L8662, Sigma) was prepared. After surface antibody staining of samples, cells were resuspended in pre-warmed HBSS. Kynurenine and/or control reagents were added and samples were kept at 37 °C. The uptake was stopped after 5 min by adding 4% PFA for 30 min at room temperature, in the dark. Samples were measured at Canto II (BD, 405 nm laser, 450/50 BP filter for kynurenine fluorescence detection).
3
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.
4
Arginine-free Complete DMEM and RPMI Preparation
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Complete Dulbecco’s Modified Eagles Media (C-DMEM) was prepared by adding bovine calf serum (SH30073.03, Thermo Fisher Scientific) to 10%, and penicillin/streptomycin (15140-122, Gibco, Life Technologies) to 1% in standard DMEM (10-013-CV, Cellgro, Corning Life Sciences). L-arginine-free (R-free) C-DMEM lacking phenol red was prepared by adding dialyzed fetal bovine serum (35-071-CV, Cellgro, Corning Life Sciences) to 10% in SILAC DMEM (D9443, Sigma-Aldrich) plus L-glutamine (584 mg/L, 25030-081, Life Technologies), L-lysine-HCl (146.2 mg/L, L8662, Sigma-Aldrich), L-leucine (52.5 mg/L, L8912, Sigma-Aldrich), sodium pyruvate (110 mg/L, 11360-070, Life Technologies), D-glucose (4500 mg/L, G8796, Sigma-Aldrich) and phenol red (15 mg/L p3532, Sigma-Aldrich). For luminescence experiments, phenol red was excluded from cell culture media. L-arginine (A8094, Sigma-Aldrich), L-citrulline (C7629, Sigma-Aldrich), and L-arginine · HCl (A5131, Sigma-Aldrich) were prepared at a stock concentration of 100 mM in sterile water. L-arginine and L-citrulline stocks were added to R-free C-DMEM to concentrations noted in the text. Complete Roswell Park Memorial Institute 1640, (C- RPMI) was prepared by adding dialyzed fetal bovine serum to 10% and penicillin/streptomycin to 1% in standard RPMI 1640 (10-040-CV, Corning Life Sciences).
5
RPMI 1640 Arginine and Citrulline Supplementation
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Complete RPMI 1640 (C-RPMI, 1.14 mM l -arginine, no l -citrulline, 10-040-CV, Corning Cellgro) was prepared by addition of 10% fetal bovine serum (SH30071, GE Healthcare) and 1% penicillin/streptomycin (15140-122, Gibco, Life Technologies). l -a rginine-free C-RPMI 1640 (R-free RPMI, 89984, Thermo Scientific) was supplemented with 10% dialyzed fetal bovine serum (35-071-CV, Cellgro, Corning Life Sciences), 1% penicillin/streptomycin (15140-122, Gibco, Life Technologies), and l -lysine (L8662, Sigma-Aldrich) to match the formulation of C-RPMI. R-free C-RPMI was supplemented with l -arginine (A8094, Sigma-Aldrich) or l -citrulline (C7629, Sigma-Aldrich) prepared in sterile water (100 mM stock) at 1 mM final concentrations.
6
Quantitative Proteomics of SMN and Gemin5 Complexes
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Control, shSMN, and shGemin5 cells were grown in DMEM containing 10% dialyzed FCS and light amino acids L-8662 ([12C]lysine; Sigma-Aldrich) and A-6969 ([12C]arginine; Sigma-Aldrich). After efficient knockdown using doxycycline induction, the control cells were grown in DMEM containing heavy isotope amino acids CNLM-291-H ([13C], [15N]lysine; Cambridge Isotope Laboratories) and CNLM-539-H ([13C], [15N]arginine; Cambridge Isotope Laboratories), whereas the SMN or Gemin5 knockdown cells were grown in medium heavy amino acids DLM-2640 ([2H]lysine; Cambridge Isotope Laboratories) and CLM-2265-H ([13C]arginine; Cambridge Isotope Laboratories) for 24 h. Next, cells were lysed in lysis buffer (50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1% NP-40, 2.5 mM MgCl2, and protease inhibitor cocktail). After incubation on ice for 10 min, the cells were lysed using 26G needle followed by mild water bath sonication to ensure complete nuclear lysis of the cells and centrifugation at 13,200 rpm for 20 min to remove cell debris. Heavy and medium pulse-labeled cell lysates were mixed in a 1:1 ratio, based on whole protein content estimated by Bradford assay (500-0006; Bio-Rad Laboratories) before processing for MS. siRNA-mediated control and pICln knockdown cells were processed similarly after the transfections.
7
Antibodies for Protein Phosphorylation Analysis
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The individual AA (l-His, l-Lys, l-Met, and l-Leu, catalog no. H5659, L8662, M5308, and L8912, respectively) were purchased from Sigma-Aldrich (Shanghai Trading Co. Ltd., Shanghai, China) . The total and site-specific phosphorylated antibodies against mTOR (Ser 2481 , catalog no. 2913/YP1134), 4EBP1 (Thr 37 , catalog no. 0018/YP0001), RPS6 (Ser 235/236 , catalog no. 4139/YP0832), and eIF2α (Ser 51 , catalog no. 1507/ YP0093) were purchased from Immuno Way (Immuno Way Biotechnology Company, Barksdale Professional Center, Newark, DE). The total and site-specific phosphorylated antibodies against Raptor (Ser792, catalog no. 2280/2083), S6K1 (Thr389, catalog no. 9202/9205), eEF2 (Thr56, catalog no. 2332/2331), and G protein β subunit-like (GβL, catalog no. 3274) were purchased from Cell Signaling Technology Inc., (Danvers, MA). The β-CN antibody (catalog no. orb18512) was purchased from Biorbyt Ltd. (Cambridge, UK) and the antibody against β-actin (catalog no. ab8226) used as a loading control was purchased from Abcam (Shanghai Trading Co. Ltd.) .
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