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MB Sample ID: SA206965
Local Sample ID: | 190905_19_0009_UCLM_CCM_023 |
Subject ID: | SU002243 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Male |
Cell Biosource Or Supplier: | CLS |
Cell Strain Details: | In vitro spontaneously transformed keratinocytes from histologically normal skin |
Cell Primary Immortalized: | HaCaT |
Cell Passage Number: | Up to the 15th passage |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002243 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Male |
Cell Biosource Or Supplier: | CLS |
Cell Strain Details: | In vitro spontaneously transformed keratinocytes from histologically normal skin |
Cell Primary Immortalized: | HaCaT |
Cell Passage Number: | Up to the 15th passage |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
190905_19_0009_UCLM_CCM_023 | SA206965 | FL025349 | Week | Exposure time |
190905_19_0009_UCLM_CCM_023 | SA206965 | FL025349 | GO g | Nanomaterial |
190905_19_0009_UCLM_CCM_023 | SA206965 | FL025349 | N1 | Replicate |
Collection:
Collection ID: | CO002236 |
Collection Summary: | HaCaT cells treated with GO1, GO2 and FLG |
Sample Type: | Keratinocytes |
Storage Conditions: | -80℃ |
Treatment:
Treatment ID: | TR002255 |
Treatment Summary: | Cells were treated with 5 ug/mL of the different GRMs for 7 days and 30 days (1 treatment/week) |
Sample Preparation:
Sampleprep ID: | SP002249 |
Sampleprep Summary: | Keratinocytes were defrosted on ice and proteins were precipitated from the lysed cell samples by adding the extraction solvent spiked with metabolites not detected in unspiked cell extracts. These metabolites, considered as internal standards, were tryptophan-d5, Anthranilic acid-(ring-13C6), Phenylthiohydantoin (PTH)-valine, Glycocholic-2,2,4,4-d4 acid (Sigma Aldrich). Then, cell extracts were incubated at -20 ˚C for 1 hour and after that, samples were vortexed and centrifuged at 18,000 x g for 10 minutes at 4 ºC. Supernatants were collected and kept on ice. A second extraction was performed from the remaining pellets, following the same steps described above. Supernatants obtained from the second extraction were collected and put together with the supernatants of the first extraction. Finally, these supernatants were dried under vacuum, reconstituted in water, resuspended with agitation for 10 minutes, centrifuged at 18,000 x g for 5 minutes at 4ºC, and transferred to vials for UHPLC-MS analysis. |
Combined analysis:
Analysis ID | AN003532 |
---|---|
Analysis type | MS |
Chromatography type | Ion pair |
Chromatography system | Waters Acquity H-Class |
Column | Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um) |
MS Type | ESI |
MS instrument type | Time of flight |
MS instrument name | Waters LCT premier |
Ion Mode | NEGATIVE |
Units | Log2 (fold-change) |
Chromatography:
Chromatography ID: | CH002609 |
Chromatography Summary: | Chromatography was performed on a 1.0-mm internal diameter x 150 mm Acquity HSS T3 1.7 µm column (Waters Corp., Milford, MA) using an ACQUITY UPLC system (Waters Corp.). The column was maintained at 40 ºC. Samples (2µL) were injected onto the column at a flow rate of 100 µL/min, for a total run time of 25 min. The following linear elution gradient was used: 100% solvent A (10mM tributylamine + 15mM acetic acid + 0.2% methanol in water), to which solvent B (methanol) was added incrementally to reach a concentration of 4% B after 1.5 min, increasing to 20% B at 3 min, 25% B at 8 min, 50% B at 10 min, 55% at 15 min, and 100% B over the next 5 min, and returning to the initial composition over the final 5 min. |
Instrument Name: | Waters Acquity H-Class |
Column Name: | Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um) |
Chromatography Type: | Ion pair |
MS:
MS ID: | MS003290 |
Analysis ID: | AN003532 |
Instrument Name: | Waters LCT premier |
Instrument Type: | Time of flight |
MS Type: | ESI |
MS Comments: | The eluent was introduced into the mass spectrometer (LCT Premier (Waters Corp.) by electrospray ionization, with capillary and cone voltages set in the negative ion mode to 2800 and 100 V, respectively. The nebulization gas was set to 600 L/hour and 300ºC. The cone gas was fixed at 50 L/hour, and the source temperature was maintained at 120ºC. Centroid data were acquired over the mass range of 50-1000 Da, using an accumulation time of 0.2 seconds per spectrum. All spectra were mass corrected in real time by reference to leucine enkephalin, infused at 10 µL/minute through an independent reference electrospray, sampled every 10 seconds. |
Ion Mode: | NEGATIVE |