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MB Sample ID: SA217829
| Local Sample ID: | SPT_C133W_1h_01311114 |
| Subject ID: | SU002360 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | ATCC, CCL-247 |
| Cell Strain Details: | HCT116 |
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Collection:
| Collection ID: | CO002353 |
| Collection Summary: | For tracing studies with [U-13C16]palmitate, HCT116 cells were cultured in growth medium in the presence of 0.1 μg/ml doxycycline for 7 days before tracer start. Growth medium was replaced to DMEM medium containing 1% (v/v) delipidated FBS 24h prior tracer start and medium exchange again 1h prior tracer trace. [U-13C16]palmitate was noncovalently bound to fatty acid-free BSA and added to culture medium at 5% of the final volume (50 μM final concentration). Media was prewarmed to 37oC in a cell incubator with 5% CO2 and cells were traced for 15 min, 1 h, and 4 h. For targeted sphingolipid analysis, cells were washed with 0.9% (w/v) NaCl and extracted with 0.25 mL of −20°C methanol, 0.25 mL -20°C chloroform, and 0.1 mL of water. The tubes were vortexed for 5 min, centrifuged at 20,000 x g at 4°C for 5 min, and the lower organic phase was collected. The remaining polar phase was extracted with 2 μL formic acid and 0.25 mL of -20 °C chloroform. The organic phases were combined, dried under air, resuspended in 80 μL Buffer B (0.2% formic acid and 1 mM ammonium formate in methanol), sonicated for 10 min, and centrifuged for 10 min at 20,000 x g at 4 °C. The supernatant was transferred to LC vials containing glass inserts for analysis on a Q-Exactive LC-MS system. |
| Sample Type: | Cultured cells |
