Immunoflourescence and mRNA analysis of human embryonic stem cells (hESCs) grown under feeder-free conditions.
This chapter describes the procedures in order to do immunofluorescence (IF) microscopy and quantitative PCR (qPCR) analysis of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol outlining the steps from initially growing the cells, passaging onto 16-well glass chambers, and continuing with the general IF and qPCR steps will be provided. The techniques will be illustrated with new results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, and PDGF signaling pathways to primary cilia in stem cell maintenance and differentiation. Furthermore, a sample qPCR experiment will be shown illustrating that these techniques can be important tools in answering basic questions about hESC biology.
Awan
Aashir
A
Laboratory for Reproductive Biology, University Hospital of Copenhagen, Copenhagen, Denmark.
Oliveri
Roberto S
RS
Jensen
Pernille L
PL
Christensen
Søren T
ST
Andersen
Claus Yding
CY
D001483:Base Sequence; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000235:genetics; D049109:Cell Proliferation; D002923:Cilia / Q000378:metabolism / Q000648:ultrastructure; D017931:DNA Primers / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D005819:Genetic Markers; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction
D015415:Biomarkers; D017931:DNA Primers; D005819:Genetic Markers; D012333:RNA, Messenger