Metabolomics Workbench : NIH Data Repository

Return to study ST002080 main page

MB Sample ID: SA196524

Local Sample ID:	RT_1
Subject ID:	SU002164
Subject Type:	Cultured cells
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116
Cell Biosource Or Supplier:	American Type Culture Collection (ATCC)
Cell Strain Details:	H9c2(2-1) is a subclone of the original clonal cell line derived from embryonic BD1X rat heart tissue by B. Kimes and B. Brandt and exhibits many of the properties of skeletal muscle.
Cell Passage Number:	3-10
Cell Counts:	80-90% confluency

Select appropriate tab below to view additional metadata details:

Sample Preparation:

Sampleprep ID:	SP002170
Sampleprep Summary:	Metabolites were extracted as previously described using 1 mL of cold MeOH/H2O (85:15). Samples were sonicated for 15 s (3×) on ice and centrifuged at 1400 rpm × 10 min at 4 ◦C (Rotor model: Eppendorf, FA-45-30-11). For protein quantification, the pellets were dried for 15 min in a rotary vacuum evaporator, then resuspended in 60 µL of denaturation buffer and sonicated for 1 min. All samples were centrifuged for 10 min at the maximum speed and the supernatant was used for protein quantification using the Bradford method. Supernatants were collected and evaporated to dryness using a SpeedVac (Savant AS160, Farmingdale, NY, USA). The metabolite samples were first derivatized through methoxyamination by adding 30 µL of 20 mg/mL solution of methoxyamine hydrochloride Antioxidants 2022, 11, 278 4 of 16 in pyridine (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37 ◦C for 2 h. Afterward, trimethylsilylation was performed by adding 30 µL of N-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide (MTBSTFA + 1% TBDMSCl; Sigma-Aldrich, St. Louis, MO, USA) and incubated for 1 h at 65 ◦C. The reaction mixture was centrifuged at 1300 rpm × 10 min at RT. Supernatants were transferred to analytical vials. Then, 20 µL per sample was added to glass vials with inserts followed by the addition of 1 mM 2-fluobiphenyl (Sigma-Aldrich, St. Louis, MO, USA) as an internal standard. Samples were processed by GC-MS-QP2010 (Shimadzu Scientific Instruments, Inc., Columbia, MD) using analytical conditions as previously described.

Sampleprep ID:

SP002170

Sampleprep Summary:

Metabolites were extracted as previously described using 1 mL of cold MeOH/H2O (85:15). Samples were sonicated for 15 s (3×) on ice and centrifuged at 1400 rpm × 10 min at 4 ◦C (Rotor model: Eppendorf, FA-45-30-11). For protein quantification, the pellets were dried for 15 min in a rotary vacuum evaporator, then resuspended in 60 µL of denaturation buffer and sonicated for 1 min. All samples were centrifuged for 10 min at the maximum speed and the supernatant was used for protein quantification using the Bradford method. Supernatants were collected and evaporated to dryness using a SpeedVac (Savant AS160, Farmingdale, NY, USA). The metabolite samples were first derivatized through methoxyamination by adding 30 µL of 20 mg/mL solution of methoxyamine hydrochloride Antioxidants 2022, 11, 278 4 of 16 in pyridine (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37 ◦C for 2 h. Afterward, trimethylsilylation was performed by adding 30 µL of N-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide (MTBSTFA + 1% TBDMSCl; Sigma-Aldrich, St. Louis, MO, USA) and incubated for 1 h at 65 ◦C. The reaction mixture was centrifuged at 1300 rpm × 10 min at RT. Supernatants were transferred to analytical vials. Then, 20 µL per sample was added to glass vials with inserts followed by the addition of 1 mM 2-fluobiphenyl (Sigma-Aldrich, St. Louis, MO, USA) as an internal standard. Samples were processed by GC-MS-QP2010 (Shimadzu Scientific Instruments, Inc., Columbia, MD) using analytical conditions as previously described.