Cdp choline

To evaluate the effect of CDP-choline on cardiac myocytes, 4 × 10⁴ cells were plated onto 96-well plates and incubated in the presence of 0.5, 1, 2.5, 5, 10, 20, 30, 40, 50, 75, and 100 mg/mL of CDP-choline (Ferrer Internacional, S.A., Barcelona, Spain) for 24 and 48 hrs. The number of cells was determined by using the colorimetric MTT assay. For cardioprotection assays, CDP-choline (10, 25, and 50 mg/mL) was added either 1 hr before (preconditioning) or 1 hr after (postconditioning) reperfusion. The number of viable cells was determined after 24 hrs. To assess changes in cell morphology, cells were washed twice with cold PBS and fixed in 100% ethanol overnight at room temperature. The remaining ethanol was discarded, and the cells were rehydrated with double distilled water for 5 min. The cells were stained with hematoxylin and eosin (Biocare Medical, CA, USA). Stained cells were analyzed using a light microscopy Axiostar Plus (Karl Zeiss Microscopy GmbH, Jena, Germany). To evaluate the effect of CDP-choline on oxidative stress-induced death, 5 mM H₂O₂ (Sigma Chemical Company, St. Louis, MO, USA) was added immediately after reperfusion, and the cells were further incubated for 6 hrs.

CPT activity was measured as described previously (7 (link)). Briefly, cells were lysed in 50 mM Tris-HCl buffer, pH 8.0, with an ultrasonic bath sonicator for 10 s, and the amount of protein in the lysate was measured using a BCA protein assay kit (Thermo Fisher Scientific). The assay reaction mixture (30 μl) contained 50 mM Tris-HCl buffer, pH 8.0, 20 mM MgCl₂, 1 mM EDTA, 0.003% Tween-20 (w/v), cell lysate (5 μg of protein), 20 μM CDP-choline [1,2-¹⁴C], and 1 mM 18:1 DAG (Sigma-Aldrich). After incubation at 37°C for the time indicated, the reaction was stopped by adding 300 μl of chloroform-methanol (1:1, v/v), and then 110 μl of water was added. After centrifugation at 12,000 g for 5 min, the organic phase was applied to a TLC plate (Merck, Darmstadt, Germany), which was developed with chloroform-methanol-water (65:25:4, v/v/v). Radiolabeled PC was analyzed using a FLA-7000 imaging analyzer and quantified using ImageQuant TL, version 8.1 (GE Healthcare UK Ltd, Amersham, UK).