4.4. Lipid Extraction

Lipids were extracted using the Bligh and Dyer method [64] with minor modifications. Lipids were extracted from cellular pellets (cell samples) or freeze-dried EVs (EV samples), respectively, by 1 mL of extraction solvent (methanol/chloroform/water, 10:5:3, v/v/v) after supplementing 20 µL of internal standard mix A (SM d18:1–17:0 and Cer d18:1–17:0, 5 µM; LPC 17:0, 25 µM; monoacylglycerol (MG) 17:0, DG 12:0–12:0, and TG 17:0–17:0–17:0, 50 µM; HexCer d18:1–12:0, 155 µM; and FA 17:0, LPE 17:1, and PC 17:0–17:0, 500 µM) and 10 µL of internal standard mix B (PE 17:0–17:0, 200 µM; CE 17:0, 250 µM; PG 17:0–17:0, 500 µM; PA 17:0–17:0, 700 µM; and PS 17:0–17:0, 2,000 µM). Four lipid classes, including PC (O) and/or PC (P), PE (P), PI, and cholesterol, could not prepare appropriate internal standards. Therefore, semi-quantitative values for each of the four lipid classes were calculated based on an external calibration comparing the ionization and fragmentation efficiencies of each available standard (PC O-16:0-18:1 and PC P-18:0-20:4, PE P-18:0-20:4, PI 18:0-20:4, and cholesterol) with each alternative internal standard (LPC 17:0, LPE 17:1, PS 17:0-17:0, and CE 17:0) (Table S1). Samples were mixed vigorously by vortex for 1 min followed by 5 min of sonication. The supernatant (700 µL) centrifugated by 16,000× g, 5 min at 4 °C was transferred to clean tubes. After mixing with 195 µL of chloroform and 195 µL of distilled water, the aqueous and organic layers were separated by vortex and the following centrifugation. The bottom layer was diluted into a half by methanol and used for lipidomic analysis. A reference sample (120 µL) was firstly prepared by mixing equal amounts (10 µL each) of 12 TNBC (6 D3H1 and 6 D3H2LN) cell extracts, which was subsequently analyzed using the in-house lipid MRM library [41].