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Inexpensive computer imaging technology was used to assess levels of insulin-like growth factor-I (IGF-I) on dot blots (DB) and alpha-Actinin on Western blots (WB). In the first procedure, known IGF-I samples were dotted on nitrocellulose membranes using a vacuum manifold. After the DB were developed and dried, the images were digitized using an HP Deskscan II flat bed scanner, exported into Image-Pro Plus and analyzed by taking the combined mean of 45 degrees and 135 degrees sample lines drawn through each dot. Dot blots corresponding to a linear concentration range from 10 to 300 ng IGF-I were assessed by this method. In the second procedure, WB were scanned with a ScanJet 3c flat bed scanner and their backgrounds were clarified using Image-Pro Plus. A second image analysis program, Alpha Imager 2000, was then used to define the boundaries of protein bands, assess pixel number and density, and to obtain final numerical data for quantifying alpha-Actinin on the WB. Collectively, the results of these two studies suggest that specific proteins may be evaluated by using relatively inexpensive image analysis software systems via pixel quantification of electronic images.
Inexpensive computer imaging technology was used to assess levels of insulin-like growth factor-I (IGF-I) on dot blots (DB) and alpha-Actinin on Western blots (WB). In the first procedure, known IGF-I samples were dotted on nitrocellulose membranes using a vacuum manifold. After the DB were developed and dried, the images were digitized using an HP Deskscan II flat bed scanner, exported into Image-Pro Plus and analyzed by taking the combined mean of 45 degrees and 135 degrees sample lines drawn through each dot. Dot blots corresponding to a linear concentration range from 10 to 300 ng IGF-I were assessed by this method. In the second procedure, WB were scanned with a ScanJet 3c flat bed scanner and their backgrounds were clarified using Image-Pro Plus. A second image analysis program, Alpha Imager 2000, was then used to define the boundaries of protein bands, assess pixel number and density, and to obtain final numerical data for quantifying alpha-Actinin on the WB. Collectively, the results of these two studies suggest that specific proteins may be evaluated by using relatively inexpensive image analysis software systems via pixel quantification of electronic images.
Background: Progeny adipofibroblast cells, derived from mature bovine adipocytes, were used to determine their ability to redifferentiate into lipid-assimilating adipocytes. Methods: Traditional cell biology methods were used, including the expression of adipogenic markers such as peroxisome proliferator-activated receptor γ (PPARγ). Results: When exposed to medium supplemented with fetal bovine serum, but not horse serum, cells began to form structures reminiscent of foci. Horse serum-supplemented medium resulted in a slowed progression towards cell conversion to lipid-assimilating adipocytes. When analyzed, horse serum was found to contain more cortisol and insulin-like growth factor-1 as well as differing fatty acid ratios. Histological observations of the horse serum-treated cultures (alone), cultures treated with a traditional differentiation induction medium (dexamethasone, methylisobutylxanthine and insulin), treated with insulin with or without different lipid compounds, or treated with a PPARγ agonist (rosiglitazone) resulted in the presence of intracellular vesicles, of which some contained lipid and some did not. Vesicles that did not stain for lipid did not possess glycogen or other types of storage moieties even though the cells expressed cellular markers thereby deeming them to be differentiated adipocytes (PPARγ protein and mRNA were expressed by cells possessing vesicles as were hormone-sensitive lipase and lipoprotein lipase proteins). Non-lipid-filled intracellular vesicle walls possessed the structural protein perilipin. Conclusion: These results are supportive of the progeny adipofibroblasts representing a unique adipogenic model that displays protracted adipogenesis.
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