CD8+ T cells and fatty acids orchestrate tumor ferroptosis and immunity via ACSL4
Description
IFNgamma reprograms ACSL4 associated phospholipids to induce tumor ferroptosis
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Steps to reproduce
Acsl4+/+ and Acsl4-/- Yumm5.2 cells or mouse T cells were given different treatments were re-suspended in 500 µl 25 mM HEPES (pH 7.4) containing 200 μM DTPA, and lysed by sonication on ice. Then, 500 µl methanol (high performance liquid chromatography, HPLC grade) was added. Samples were immediately frozen and kept at −80 °C. Upon thawing, after addition of an internal standard mix (EquiSplash; Avanti Polar Lipids), lipids were extracted using the Bligh & Dyer method. The organic phase was dried using a SpeedVac concentrator (Thermo Scientific) and resuspended in 100 µL of mobile phase A (hexanes/isopropanol 30:40 v/v) for injection into the liquid chromatography with tandem mass spectrometry (LC-MS/MS) system (Exion LC coupled to Triple Quadrupole 6500+, Sciex). Phospholipids were separated by class upon normal-phase HPLC using a gradient (25 % to 95 %) with mobile phase B (hexanes/isopropanol/water 30:40:7 v/v/v, 5 mM ammonium acetate) on a HILIC column (Kinetex 2.6 µm, 100 Å, 100 x2 .1 mm; Phenomenex). Ions were analyzed by multiple-reaction monitoring of phospholipid molecular species, containing d0- and-d5-arachidonoyl chains in combination with common acyl chains: 16:0, 16:1, 18:0, 18:1, 18:2, 20:0, 20:2, 20:4, 20:5, 22:0, 22:2, 22:4, 22:5, and 22:6. The m/z transitions monitored were those from the deprotonated molecular negative ion (or the acetate adduct negative ion in the case of phosphatidylcholine species) to the corresponding acyl ion fragments. The chromatographic peaks (ion count vs time) for each analyte and internal standard were integrated using MultiQuant software (Sciex). Analyte relative abundances were calculated as the area ratio of each to the corresponding internal standard in their class.