Zorbax sil column

We dissolved 2000 pmol of neurosporaxanthin in acetone and mixed it with reaction buffer (20 mM Tricine, 150 mM NaCl, 0.5 mM TCEP, 0.2% Triton x-100 at pH 7.4). Then 50 μg of enzyme solution was added to the mixture and incubated at 37 °C under shaking at 600 rpm in a thermomixer (Eppendorf, Hamburg, Germany) for 10 min. The reactions were stopped by the addition of 100 μL of 10% acetic acid in water (v/v), 400 μL of acetone, 400 μL of diethyl ether, and 100 μL of petroleum ether. Organic and aqueous layers were separated, collected, and evaporated in a dry vacuum centrifuge (Eppendorf). Carotenoids and retinoids were analyzed by HPLC using a Zorbax Sil column (Agilent Technologies), as outlined above.

Following treatments as indicated in the text, monocytes or MΦ were incubated with radiolabeled ³[H]-25D₃ (PerkinElmer, Waltham, MA) substrate for 5 hours in serum-free RPMI media. [³H]-metabolites were purified using a C18 column and separated using a Zorbax-sil column (Agilent, Santa Clara, CA). Radioactivity was measured in each sample by scintillation counting. The amount of each metabolite present was quantified from counts per minute plotted against elution profiles of standards for 25D₃, 1,25D₃, and 24,25D₃ [12 (link)].