Cell culture and pulse labeling (remodeling)

Jurkat cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 10 mM HEPES, d-glucose (to 25 mM) and 1 mM sodium pyruvate at 37°C in a 5% CO₂ atmosphere. For FA incorporation studies, Jurkat cells were incubated in the presence of 20 μM AA or 20 μM AA-alk for 2 h at 37°C. Cells were then washed twice with culture medium, and cellular lipids were extracted in chloroform using heptadecanoyl-PC as an internal standard (30). For pulse-labeling experiments, Jurkat cells (6 × 10⁷) were pulse labeled in 3 ml of culture medium (2% FBS) containing 20 μM [³H]AA or 20 μM AA-alk for 2 h at 37°C. Cells were then washed twice with culture medium and incubated for another 0, 4, or 24 h before cellular lipid extraction. Cellular lipids were extracted in chloroform (30), PL classes were separated by reverse phase-HPLC (RP-HPLC) (31), and fractions containing neutral lipids, PE, PI, phosphatidylserine (PS), and PC were collected using elution times determined with PL standards. The internal standard heptadecanoyl-PC was added to each fraction prior to further analyses.