title: [The establishment of "two-step sequential filtration method" on the yield rate of purified islets in rats].
abstract: OBJECTIVE
  To develop a simple, rapid and reliable method of purifying Sprague-Dawley (SD) rat islets by sequential filtration through two cell strainers of different sizes and to evaluate the efficacy of the method.
  
  
  METHODS
  Islets were isolated from 8 to 12-week-old clean grade Sprague-Dawley rat pancreases using the standard collagenase digestion procedure and purified with either the generally used Ficoll density gradient method or the innovative two-step sequential filtration method. The purity and vitality of the isolated islets were visualized and assessed with DTZ and AO/PI staining. Glucose stimulating tests were performed to assay cell activity, and immunohistochemical staining was used to evaluate the synthesis function of islet cells.
  
  
  RESULTS
  The yield of islets in the two-step filtration method group was 782±115 IEQ per rat, which was significantly higher than in the conventional Ficoll density gradient method group (598 ± 135 IEQ per rat, P < 0.01). Purity of the isolated islets in the two-step filtration method group was 90%-100% and vitality was over 95%. In the conventional Ficoll density gradient method group, islet purity was 65%-85% and vitality was 85%-95%. With regard to the high-sugar stimulation test in the two-step filtration method group, insulin concentrations in islets cultured for 24 hours were significantly higher than in those that were freshly purified (76.9 ± 6.1 μg/L vs 49.4 ± 3.9 μg/L; P < 0.01).
  
  
  CONCLUSIONS
  A two-step sequential filtration method for rat islet purification was developed and the method was simple and reliable, with high islet vitality, purity and yield.
authors.affiliation: Department of Endocrinology and Inborn Metabolic Diseases, Children's Hospital of Fudan University, Shanghai 201102, China. luofh@fudan.edu.cn.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Lu
authors.forename: Shui-Xian
authors.initials: SX
authors.lastname: Shen
authors.forename: Di-Jing
authors.initials: DJ
authors.lastname: Zhi
authors.forename: Fei-Hong
authors.initials: FH
authors.lastname: Luo
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods*; D005260:Female; D005374:Filtration; D007150:Immunohistochemistry; D007328:Insulin / Q000096:biosynthesis; D007515:Islets of Langerhans / Q000166:cytology*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D007328:Insulin