Return to study ST003020 main page
MB Sample ID: SA327678
| Local Sample ID: | MEL_longterm_PKCi_HILIC_2000nM_FA_GFX_3 |
| Subject ID: | SU003134 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Cell Strain Details: | MEL |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003134 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Cell Strain Details: | MEL |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| MEL_longterm_PKCi_HILIC_2000nM_FA_GFX_3 | SA327678 | FL038881 | 2000nM_FA_GFX | Treatment |
Collection:
| Collection ID: | CO003127 |
| Collection Summary: | One million cells from culture were collected via centrifugation for 20 seconds at 18,000xG, washed with 0.9% NaCl, and collected via centrifugation for 20 seconds at 18,000xG |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR003143 |
| Treatment Summary: | MEL cells were cultured for 48hr in 2000 nM folic acid + vehicle, 100 nM folic acid + vehicle, 2000 nM folic acid + AICAR (500 nM), and 2000 nM folic acid + SHIN1 (1.25 uM). |
Sample Preparation:
| Sampleprep ID: | SP003140 |
| Sampleprep Summary: | Pellet was resuspended in extraction buffer (80% Methanol, 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC-MS water, supplemented with isotopically-labelled amino acid standards [Cambridge Isotope Laboratories, MSK-A2-1.2], aminopterin, and reduced glutathione standard [Cambridge Isotope Laboratories, CNLM-6245-10]). Samples were vortexed for 10 sec, then centrifuged for 10 minutes at 18,000 g to pellet cell debris. The supernatant was divided into two tubes and dried on ice using a liquid nitrogen dryer. One tube of dried sample was used for polar metabolite detection. Dried samples were resuspended in 25 μL water |
Combined analysis:
| Analysis ID | AN004954 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish |
| Column | EMD Millipore ZIC-HILIC (100 x 2.1 mm,3.5 um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | Arbitrary Units |
Chromatography:
| Chromatography ID: | CH003738 |
| Chromatography Summary: | Sample was injected into a ZIC-pHILIC 150 x 2.1 mm (5 μm particle size) column (EMD Millipore). operated on a Vanquish™ Flex UHPLC Systems (Thermo Fisher Scientific, San Jose, CA, USA). Chromatographic separation was achieved using the following conditions: buffer A was acetonitrile; buffer B was 20 mM ammonium carbonate, 0.1% ammonium hydroxide in water; resulting pH is around 9 without pH adjustment. Gradient conditions we used were: linear gradient from 20% to 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B at 150 mL/min flow rate. The column oven and autosampler tray were held at 25 °C and 4 °C, respectively. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | EMD Millipore ZIC-HILIC (100 x 2.1 mm,3.5 um) |
| Column Temperature: | 25 |
| Flow Gradient: | linear gradient from 20% to 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B at 150 mL/min flow rate |
| Flow Rate: | 150 mL/min |
| Solvent A: | 100% acetonitrile |
| Solvent B: | 100% water; 20 mM ammonium carbonate; 0.1% ammonium hydroxide |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004694 |
| Analysis ID: | AN004954 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Polar metabolites were relatively quantified while referencing an in-house library of chemical standards and using TraceFinder 4.1 (Thermo Fisher Scientific, Waltham, MA, USA), with a 5 ppm mass tolerance. |
| Ion Mode: | UNSPECIFIED |
