Related Experiment Video

Updated: Apr 22, 2026

Using Primary Neurosphere Cultures to Study Primary Cilia
08:14

Using Primary Neurosphere Cultures to Study Primary Cilia

Published on: April 14, 2017

8.6K

Immunofluorescence Microscopy and mRNA Analysis of Human Embryonic Stem Cells (hESCs) Including Primary Cilia

Maj Linea Vestergaard1, Aashir Awan, Caroline Becker Warzecha

  • 1Laboratory of Reproductive Biology, Section 5712, University Hospital of Copenhagen, Copenhagen OE, Denmark, maj.linea.vestergaard@rh.regionh.dk.

Methods in Molecular Biology (Clifton, N.J.)
|October 12, 2014
PubMed

Insights

This study details immunofluorescence microscopy and quantitative PCR protocols for feeder-free human embryonic stem cells. These methods analyze signaling pathways and transcriptional factors crucial for stem cell maintenance and differentiation.

Area of Science:

  • Stem Cell Biology
  • Molecular Biology
  • Cellular Imaging

Background:

  • Human embryonic stem cells (hESCs) require specific culture conditions for maintenance and differentiation.
  • Understanding signaling pathways is crucial for controlling hESC fate.
  • Feeder-free culture systems offer advantages for hESC research.

Purpose of the Study:

  • To provide detailed protocols for immunofluorescence microscopy (IFM) and quantitative PCR (qPCR) in hESCs.
  • To demonstrate the application of IFM and qPCR in analyzing signaling pathways and transcriptional factors.
  • To highlight the utility of these techniques in hESC research.

Main Methods:

  • Established protocols for culturing hESCs under feeder-free conditions.
  • Detailed procedures for IFM to determine cellular localization of proteins.
  • Comprehensive qPCR methods for analyzing gene expression.

Main Results:

  • IFM successfully localized transcriptional factors and signaling pathway components (Hedgehog, Wnt, PDGF, TGFβ) to primary cilia.
  • qPCR experiments demonstrated the ability to assess gene expression related to stem cell biology.
  • The combined techniques provide insights into hESC maintenance and differentiation.

Conclusions:

  • IFM and qPCR are robust techniques for analyzing hESC biology under feeder-free conditions.
  • These methods facilitate the study of key signaling pathways and transcriptional regulation in hESCs.
  • The described protocols are valuable tools for basic hESC research and potential therapeutic applications.

More Related Videos

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development14:19

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Published on: March 25, 2022

3.3K
Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR09:03

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Published on: May 29, 2014

11.1K

Related Concept Videos

Microtubules in Signaling01:22

Microtubules in Signaling

The primary cilium, made up of microtubules, acts as antennae on the cell surfaces for relaying external stimuli into the cells. These fine hair-like structures are present, generally one per cell. These are non-motile cilia in a 9+0 microtubules arrangement, where the central pair of microtubules are absent. The primary cilia arise from the basal body embedded in the cell membrane. Intraflagellar transport (IFT) carries requisite proteins from the cytoplasm to the cilium because the primary...
1.5K