Unicel dxh 800 coulter cellular analysis system

Venous blood samples were collected in tubes containing dipotassium ethylenediaminetetraacetic acid. Hematocrit levels were determined with the Beckman Coulter UniCel DxH 800 Coulter Cellular Analysis System (Indianapolis, IN, USA) for all samples. Afterwards, the erythrocytes were separated from the plasma by centrifugation (3000 rpm, 1500g, for 10 min) and washed with an equal volume of saline. After the removal of the saline, the cells were resuspended with saline to a hematocrit of 45%. These erythrocyte suspensions and plasma were stored at −80°C in Eppendorf vials freshly treated with butylated hydroxytoluene, as previously described [20] (link).
Erythrocyte suspensions were thawed before sample preparation, and 200 μL of erythrocyte suspension and 2 mL of 3N methanolic hydrochloric acid including internal standard—heptadecanoic acid (C17:0) at 0.1-μM concentration—were added to the 13 mL screw-cap glass tubes. Transmethylation was performed at 90°C for 3 h. After the tubes were cooled to room temperature, 2 mL of hexane was added and the tubes were closed again and vortexed for 10 s. The upper phase was transferred to a clean glass tube and evaporated in the SpeedVac (scan speed 32, 1900 rpm, 25°C). Finally, the residue was resuspended with 100 μL of hexane and the sample was transferred to a gas chromatography injection vial with a screw cap [9 (link),20 (link)].