title: Immunofluorescence Microscopy and mRNA Analysis of Human Embryonic Stem Cells (hESCs) Including Primary Cilia Associated Signaling Pathways.
abstract: This chapter describes the procedures for immunofluorescence microscopy (IFM) and quantitative PCR (qPCR) analyses of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol is provided outlining the steps from initially growing the cells, passaging onto 16-well glass chambers, and continuing with the general IFM and qPCR anlysis. The techniques are illustrated with results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, PDGF, and TGFβ signaling pathways to primary cilia in stem cell maintenance and differentiation. Furthermore, a sample qPCR experiment is experimentally shown illustrating that these techniques can be important tools in answering basic questions about hESC biology.
authors.affiliation: Laboratory of Reproductive Biology, Section 5712, University Hospital of Copenhagen, Copenhagen OE, Denmark, maj.linea.vestergaard@rh.regionh.dk.
authors.forename: Maj Linea
authors.initials: ML
authors.lastname: Vestergaard
authors.forename: Aashir
authors.initials: A
authors.lastname: Awan
authors.forename: Caroline Becker
authors.initials: CB
authors.lastname: Warzecha
authors.forename: Søren Tvorup
authors.initials: ST
authors.lastname: Christensen
authors.forename: Claus Yding
authors.initials: CY
authors.lastname: Andersen
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D048430:Cell Shape; D002478:Cells, Cultured; D002923:Cilia / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D061252:Feeder Cells / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology; D000066449:Human Embryonic Stem Cells / Q000378:metabolism*; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics*
chemical_list: D015415:Biomarkers; D012333:RNA, Messenger