NDLI: Human ES cells: starting culture from frozen cells.

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Year: 2006 Issue: 2

Year: 2006 Issue: 1

ES cell-derived neuroepithelial cell cultures.

Human ES cells: starting culture from frozen cells.

Assay for adhesion and agar invasion in S. cerevisiae.

Dissection of Drosophila ovaries.

Propagation of human embryonic stem (ES) cells.

Nuclear transfer into mouse oocytes.

Dissection of larval CNS in Drosophila melanogaster.

A rapid technique for the visualization of live immobilized yeast cells.

Passaging HuES human embryonic stem cell-lines with trypsin.

Freezing human ES cells.

Long-term imaging mammalian cells using wide-field microscopy.

Gene-gun transfection of hippocampal neurons.

Horizontal slice preparation of the retina.

Gibberella zeae ascospore production and collection for microarray experiments.

Monitoring actin disassembly with time-lapse microscopy.

Light/dark transition test for mice.

Measuring exocytosis in neurons using FM labeling.

Human ES cells: starting culture from frozen cells.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Trish, Erin Dimos, John Eggan, Kevin
Description	Country affiliation: United States Author Affiliation: Trish E ( Dept of Molecular and Cellular Biology, Harvard University, USA. etrish@mcb.harvard.edu)
Abstract	Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock. First, a one to two day old ten cm plate of approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator. A frozen vial of cells from long term liquid nitrogen storage or a -80 C freezer is sourced and quickly submerged in a 37 C water bath for quick thawing. Cells in freezing media are then removed from the vial and placed in a large volume of HuES media. The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate. Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate. It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate. The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.
File Format	HTM / HTML
e-ISSN	1940087X
DOI	10.3791/86
Journal	Journal of Visualized Experiments
Issue Number	1
Language	English
Publisher	MyJove Corp.
Publisher Date	2006-11-09
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Cell Culture Techniques Embryonic Stem Cells Cytology Freezing Animals Cells, Cultured Culture Media Embryo, Mammalian Fibroblasts Mice Video-audio Media
Content Type	Text
Resource Type	Article

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