Glycerol-3-Phosphate dehydrogenase (E.C.1.1.1.8) is expressed in cultured chicken embryonic adipofibroblasts and upregulated by embryonic chicken serum.

Chicken embryonic adipofibroblasts (CEA) accumulate intracytoplasmic lipids when cultured in medium containing chicken serum (CS), but not in medium with fetal bovine serum (FBS). To characterize this process of lipid accumulation, we evaluated the expression of the enzyme glycerol-3-phosphate dehydrogenase (E.C.1.1.1.8) (GPDH), first in chicken tissues and then in CEA cultured under diverse conditions. GPDH activity in adipose depots from 4-wk-old broiler chickens was similar or higher than that shown by liver, the main organ for fatty acid synthesis in chickens, while skeletal muscle had the lowest levels of GPDH. In vitro, GPDH activity increased in CEA cultured in the presence of CS but not in medium with FBS, paralleling the lipid accumulation by these cells. Both lipid accumulation and GPDH activity were further increased in CEA cultured in the presence of embryonic CS. Our results show that GPDH is highly expressed in avian tissues related to lipid metabolism and therefore can be a reliable marker for avian adipogenesis, and suggest that ECS is an optimum source for the purification of avian adipogenic factors.

Colorimetric quantitation of in vitro cell density using carmine, a chromosome-specific stain.

Cell number is usually evaluated during in vitro studies to estimate metabolic or pharmacological effects of specific compounds. However, estimation of in vitro cell density by direct cell counting is a laborious and time-consuming task, whereas indirect methods for cell quantitation have serious disadvantages such as environmental costs or inaccuracies derived from non-specific interferences. We developed a new method for in vitro cell density quantitation which employs carmine, a natural dye widely used for chromosome staining in cytological studies. Normal or transformed murine fibroblasts, avian normal fibroblasts, human epithelial HeLa cells, and insect cells, inoculated at a range of cell densities, were fixed with 4% formaldehyde/PBS and stained with 0.4% alcoholic-HCl carmine. The stain retained in cell monolayers was extracted with 0.01 M NaOH and spectrophotometrically measured at 531 nm. Invariably, high correlation coefficients between cell number and absorbance were obtained for each cell type, within a range of 5 x 10(3) to 5 x 10(5) cells. Moreover, identical cell growth curves were obtained when cell number was estimated over several days of culture by both direct cell counting and carmine staining methods. Our results show that the carmine staining method represents an easy, precise and reliable alternative for in vitro cell quantitation, avoiding interferences caused by cell components modulable by culture treatments, and over a wide range of cell types and cell densities.