[Method for the isolation of human glomerular basement membrane (author's transl)].
In order to obtain pure glomerular basement membrane for chemical analysis, we could develop the following method. The isolated glomeruli are treated with a 0.08% polyoxyethylene-sorbitaine-monooleate for 3-5 minutes and they are homogenized in this detergent. The homogenate obtained is placed in a sucrose gradient with the specific density between 1021 to 1081. After standing for 30 minutes in a refrigator, the homogenate was separated into three layers. The uppermost layer contained fragmented cell debris, the second layer was consisted of the endothelial and epithelial cells. In the third layer, which was clearly separated from upper two layers by the translucent zone, the basement membrane were found. The position of the third layer is different in normal and pathological glomeruli, and the width of the layer is very narrow. According to this method only a very little contamination with other glomerular cell fraction is observed. The application of the 0.08% polyoxyethylene-sorbitane-monooleate did not induce any appreciable change in the structure of basement membrane, because carbohydrate composition and sialic acid content of this preparation were essentially identical with the data reported by several authors who prepared basement membrane by ultrasonic method. Electron microscope also revealed that the isolated glomerular basement membrane preserved lamina densa completely attached with partly stripped lamina rara interna and externa. Our method to break glomeruli is inexpensive and less time-consuming than the breakage with ultrasonic vibration. Isolation procudure with density gradient is obviously superior to the simple centrifugation or settling method adopted by others.
D001485:Basement Membrane / Q000166:cytology*; D002458:Cell Fractionation; D002474:Cell-Free System; D003920:Diabetes Mellitus / Q000473:pathology; D004727:Endothelium; D004847:Epithelial Cells; D006801:Humans; D007678:Kidney Glomerulus / Q000166:cytology* / Q000473:pathology / Q000648:ultrastructure; D008722:Methods
Eine verbesserte Isolierungsmethode für menschliche glomeruläre Basalmembran