TY - JOUR
T1 - Immunoflourescence and mRNA analysis of human embryonic stem cells (hESCs) grown under feeder-free conditions
AU - Awan, Aashir
AU - Oliveri, Roberto S
AU - Jensen, Pernille L
AU - Christensen, Søren Tørholm
AU - Andersen, Claus Yding
PY - 2010/1/1
Y1 - 2010/1/1
N2 - 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.
AB - 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.
U2 - 10.1007/978-1-60761-369-5_11
DO - 10.1007/978-1-60761-369-5_11
M3 - Journal article
C2 - 19907979
SN - 1940-6029
VL - 584
SP - 195
EP - 210
JO - Methods in molecular biology
JF - Methods in molecular biology
ER -