title: Endothelial cell high-enrichment from endovascular biopsy sample by laser capture microdissection and fluorescence activated cell sorting.
abstract: OBJECTIVE
  Endovascular sampling and characterization from patients can provide very useful information about the pathogenesis of different vascular diseases, but it has been limited by the lack of an effective method of endothelial cell (EC) enrichment. We optimized the EC yield and enrichment from conventional guide wires by laser capture microdissection (LCM) and fluorescence activated cell sorting (FACS) technique, and addressed the feasibility of using these enriched ECs for downstream gene expression detection.
  
  
  METHODS
  Iliac artery endovascular samples from 10 patients undergoing routine catheter angiography were collected using conventional 0.038 in. J-shape guide wires. Each of these samples was equally divided into two parts, which were respectively used for EC enrichment by immunocytochemistry-coupled LCM or multiple color FACS. After RNA extraction and reverse transcription, the amplified cDNA was used for quantitative polymerase chain reaction (qPCR).
  
  
  RESULTS
  Fixed ECs, with positive CD31 or vWF fluorescent signal and endothelial like nucleus, were successfully separated by LCM and live single ECs were sorted on FACS by a seven color staining panel. EC yields by LCM and FACS were 51 ± 22 and 149 ± 56 respectively (P < 0.001). The minimum number of fixed ECs from ICC-coupled LCM for acceptable qPCR results of endothelial marker genes was 30, while acceptable qPCR results as enriched by FACS were attainable from a single live EC.
  
  
  CONCLUSIONS
  Both LCM and FACS can be used to enrich ECs from conventional guide wires and the enriched ECs can be used for downstream gene expression detection. FACS generated a higher EC yield and the sorted live ECs may be used for single cell gene expression detection.
authors.forename: Zhengda
authors.initials: Z
authors.lastname: Sun
authors.forename: Hua
authors.initials: H
authors.lastname: Su
authors.forename: Brian
authors.initials: B
authors.lastname: Long
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Sinclair
authors.forename: Steven W
authors.initials: SW
authors.lastname: Hetts
authors.forename: Randall T
authors.initials: RT
authors.lastname: Higashida
authors.forename: Christopher F
authors.initials: CF
authors.lastname: Dowd
authors.forename: Van V
authors.initials: VV
authors.lastname: Halbach
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Cooke
mesh_terms: D001706:Biopsy; D018076:DNA, Complementary / Q000235:genetics; D042783:Endothelial Cells / Q000166:cytology*; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D007083:Iliac Artery / Q000166:cytology* / Q000000981:diagnostic imaging / Q000601:surgery; D061346:Laser Capture Microdissection; D012313:RNA / Q000235:genetics; D011859:Radiography; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D018076:DNA, Complementary; D012313:RNA