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MB Sample ID: SA354192

Local Sample ID:210309_MDA_Timecourse_RSL3_n1_4h_RSL3_10uM_6
Subject ID:SU003381
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

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Subject:

Subject ID:SU003381
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
210309_MDA_Timecourse_RSL3_n1_4h_RSL3_10uM_6SA354192FL041320Breast cancer cellsSample source
210309_MDA_Timecourse_RSL3_n1_4h_RSL3_10uM_6SA354192FL04132010 µM RSL3Treatment
210309_MDA_Timecourse_RSL3_n1_4h_RSL3_10uM_6SA354192FL0413204 htime point

Collection:

Collection ID:CO003374
Collection Summary:Cultured cells were washed, trypsinized, counted and flash-frozen in liquid N2 and stored at -80°C.
Sample Type:Breast cancer cells
Storage Conditions:-80℃

Treatment:

Treatment ID:TR003390
Treatment Summary:Treatment of brest cancer cells (MDA-MB-231 cells) for the analysis of oxidized phosphatidylethanolamine (oxPE), oxidized phosphatidylcholine (oxPC), and oxidized phosphatidylinositol (oxPI): Human MDA-MB-231 breast cancer cells were treated with vehicle (DMSO) or RSL3 (1 or 10 µM) with or without ferrostatin-1 (3 µM) for 2 h, 4 h, 6 h, or 24 h or with SCs (1, 3, and 10 µM) for 2 h at 37°C and 5% CO2. Cells were harvested, washed with PBS pH 7.4, snap-frozen, and stored at -80°C.

Sample Preparation:

Sampleprep ID:SP003388
Sampleprep Summary:Phospholipids were extracted from cell pellets by successive addition of PBS pH 7.4, methanol, chloroform, and saline to a final ratio of 14:34:35:17. Evaporation of the organic layer yielded a lipid film that was dissolved in methanol and subjected to UPLC-MS/MS.
Extract Storage:-80℃

Combined analysis:

Analysis ID AN005345
Analysis type MS
Chromatography type Reversed phase
Chromatography system Waters Acquity H-Class
Column Waters ACQUITY UPLC BEH C8 (100 x 2.1mm,1.7um)
MS Type ESI
MS instrument type QTRAP
MS instrument name ABI Sciex 6500+
Ion Mode NEGATIVE
Units absolute intensities

Chromatography:

Chromatography ID:CH004047
Chromatography Summary:Chromatographic separation of phospholipids was carried out on an Acquity BEH C8 column (1.7 μm, 130 Å, 2.1×100 mm, Waters, Milford, MA) using an ExionLC UHPLC system.
Instrument Name:Waters Acquity H-Class
Column Name:Waters ACQUITY UPLC BEH C8 (100 x 2.1mm,1.7um)
Column Temperature:45°C
Flow Gradient:The gradient was ramped from 75 to 85% B over 5 min and further increased to 100% B within 2 min, followed by isocratic elution for another 2 min.
Flow Rate:0.75 ml/min
Solvent A:water/acetonitrile 90/10, 2 mM ammonium acetate
Solvent B:water/acetonitrile 5/95, 2 mM ammonium acetate
Chromatography Type:Reversed phase

MS:

MS ID:MS005075
Analysis ID:AN005345
Instrument Name:ABI Sciex 6500+
Instrument Type:QTRAP
MS Type:ESI
MS Comments:Targeted MRM with pre-optimized settings and subsequent automated integration of selected signals using Analyst 1.6.3 or Analyst 1.7.1 (Sciex). Oxidized phospholipid anions (PE and PI: [M-H]-; PC: [M+OAc]-) were detected after fragmentation to both fatty acid anions. Retention time windows for oxidized PC and oxidized PE were predicted based on the analysis of oxidized PC(16:0/20:4) (oxPAPC) (Avanti Polar Lipids, Alabaster, AL; 1[O]: 2.8–4.6 min; 2[O]: 2.9–4.3 min; 3[O]: 1.45–2.3 min). Quantitation is based on the most intense signals of oxidized fatty acid anion fragments. The fractions of phospholipids with one 1[O], two 2[O], or three oxygens 3[O] incorporated comprise multiple isomeric species that were summed and normalized to DMPC (for oxidized PC and oxidized PI) or DMPE (for oxidized PE) and cell number.
Ion Mode:NEGATIVE
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