Equisplash

Total lipid extracts from the cell pellets were prepared using MTBE liquid–liquid extraction. Briefly, 400 μl ice-cold methanol was added to the cell pellet followed by 30 s of sonication and 30 s of vortex mixing. Then, 10 μl of the internal standard (EquiSplash [#330731], Avanti Polar Lipids) was added to the samples followed by the addition of 500 μl MTBE. The mixture was incubated at 4 °C for 1 h with 650 rpm shaking. Water (500 μl) was then added followed by vortex mixing and incubated at 4 °C for 15 min with 650 rpm shaking. The samples were centrifuged at 8000g for 8 min at 4 °C to induce phase separation. The upper (organic) layer was transferred and stored on ice. MTBE (200 μl) was added to the lower (aqueous) phase followed by vortex mixing (30 s) and incubation at 4 °C for 15 min with 650 rpm shaking. The upper (organic) phase was removed and combined with the first extraction. The combined organic layers were dried with nitrogen at 30 °C, and the dried lipid extract was resuspended in 200 μl of chloroform:methanol (1:1, v/v) with 200 μM of butylated hydroxytoluene and stored at −20 °C. Prior to analysis, extracts were further diluted 1:5 in acetonitrile:isopropanol:water (1:2:1, v/v/v). The lower aqueous phase was used to determine the protein content using a BCA kit (bicinchoninic acid assay, Thermo Fisher Scientific).

Cells were cultured for 72 h in media depending on
cell type within glass culture dishes and washed with warm FBS-free
media before capillary sampling. Cells were kept in fresh FBS-free
media. The 35 mm culture dish was introduced to the Yokogawa SS2000
Single Cellome System, where living single cells were sampled using
10 μm capillaries (Yokogawa). Single cells were manually selected
at random in the direct mode with the following pressures: presampling
6 psi, sampling 14 psi, and postsampling 3 psi. The cells were sampled
with a single pick and held for 200 ms. The tips were immediately
frozen after cell sampling using dry ice. Single cells were transferred
and stored at −80 °C for future use. Cells were kept at
37 °C with 5% CO₂ during sampling.
Cells were
transferred from the capillary into total recovery LC–MS vials
(Waters) by backfilling the capillaries with 5 μL of lysis solvent
that consisted of starting mobile phase 70:30 A/B spiked with EquiSPLASH
(Avanti Polar Lipids, cat no. 330731; 16 ng/mL) and using a gas syringe
with a Luer lock adapter to elute the solution into the vial, as described
previously.^{13 (link)} LC–MS/MS (DDA lipidomics)
analysis was performed on the same day of elution, and the total 5
μL volume was injected into the column.

All standards were purchased from Avanti Polar Lipids: Ultimate SplashOne (#330820), dFA 18:1 (#861809), dFA 20:4 (#861810), dCer d18:0/13:0 (#330726), Glu Cer(d18:1-d7/15:0) (#330729), dLacCer d18:1/15:0 (#330727), 15:0-18:1-d7-PA (#791642), EquiSPLASH (#330731).