Upregulation of 12/15-LOX mRNA expression in murine peripheral monocytes by IL-4. The blood of 20 mice was pooled, and the white blood cells were prepared by density step-gradient centrifugation.15 After washing twice with PBS, the cells were plated to Petri-dishes and macrophages were allowed to adhere overnight. Nonadherent cells (mainly granulocytes and lymphocytes) were removed by washing the plates five times with culture medium. The adherent cells were scraped off the plates and were resuspended in 0.2 mL of PBS. Total RNA and RT-PCR of the GAPDH and 12/15-LOX mRNA were performed as described in Material and Methods. For quantification (lower part of the figure) the intensity of the GAPDH band obtained from IL-4–treated cells was set 100% and the intensity of the other bands were related to it. Inset, original PCR pattern obtained from the different samples.