Im 11 three dimensional mobile manipulator

A PC-10 pipette puller (Narishige Group, Tokyo, Japan) was used for preparing the borosilicate glass nanotips (length 55 mm, tip i.d. 2–6 μm, capillary i.d. 0.6 mm) according to the manufacturer’s instructions. The 1st step heating temperature was set at 60.5 °C, and the 2nd step heating temperature was set at 42−50 °C, depending on the tip diameter. The diameter of each prepared nanotip was confirmed under the microscope.
The LD sampling was conducted using an IM-11 three-dimensional mobile manipulator (Narishige Group), coupled with an IX71 inverted microscope in a bright field, as in our previous studies [24 (link),27 (link)]. The culture medium of the cells was washed and then replaced with 0.6 mL 160 mM ammonium formate solution, and the LDs were aspirated into nanotips using the microinjector. The obtained single LD was immediately extracted with 10 µL of methanol/isopropanol (1:9 v/v, with 0.1% trifluoroacetate).

LD sampling was performed according to the previously reported method [25 (link)] with slight modification. For semi-quantification of TG in LDs, nanotips were preloaded with 40 pmol of TG 11:0/11:0/11:0 (in 5 μL MeOH) as the IS, and the solvent was volatilized in nitrogen. Before LD extraction, the culture medium was discarded and replaced with 160 mM of NH₄COOH solution. The operation was aided by an IM-11 three-dimensional mobile manipulator (Narishige, Tokyo, Japan) and guided by an inverted IX71 microscope in a bright field. LDs were aspirated into nanotips, and the nanotips were then backfilled with 5 μL of organic solvent (MeOH/iPrOH = 1:9 v/v, with 0.1% trifluoroacetate). The detection was conducted on an LTQ Orbitrap mass spectrometer with a nanoESI source, of which the instrument parameters were the same as described previously [25 (link)]. For semi-quantitative comparison, the total levels of TG, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) were calculated as the sum of all the molecular species within the same class.