Stem Cell Culture Protocols

Aggregation of ES Cells with Mouse Morula- Stage Embryos Protocol Protocol for the aggregation of ES cells with mouse morula-stage embryos. Includes: Preparation of aggregation walls; ES cell preparation; Embryo preparation and aggregation.

Culture of Human Embryonic Stem Cells (hESC) Protocol Protocol for the culture of human embryonic stem cells (hESC).

Culturing Trophoblast Stem (TS) Cell Lines Protocol Culture conditions have been established for a second blastocyst-derived cell line, trophoblast stem (TS) cells, in addition to embryonic stem (ES) cells. This protocol describes a method for culturing TS cell lines. These cells can then be used to study trophoblast differentiation and placental function.

Differentiation of Embryonic Stem (ES) Cells Using the Hanging Drop Method In vitro differentiation of ES cells occurs when the cells are allowed to aggregate in suspension culture in the absence of mouse embryonic fibroblast (MEF) feeders and leukemia inhibitory factor (LIF). Hanging drops provide a uniform aggregate size, which is then expanded by continued growth in suspension culture. The embryoid bodies are then plated and allowed to differentiate further in culture.

ES / MEF Cell Culture and Electroporation of Targeting Construct Protocol Protocol for ES/MEF cell culture and electroporation of targeting construct.

Expansion of Targeted ES Clones Protocol Protocol for the expansion of targeted ES clones.

Hematopoietic Colony Forming Cell Assays Protocol Protocol for the hematopoietic colony forming cell assay.

Human Embryonic Stem Cell Protocols Human embryonic stem cells (hESCs) are pluripotent cells capable of differentiation to representatives of all three germ layers. Includes: Isolation of Primary Mouse Embryo Fibroblasts; Thawing and preparing p1 MEF feeder plates; Preparation of MEF- Conditioned Medium (MEF-CM; Microdissection Passaging of hESCs; Bulk passaging of hESC; Cryopreservation of hESCs; Thawing of hESCs; Karyotyping.

Neural Stem Cell Culture: Neurosphere Generation, Microscopical Analysis and Cryopreservation Describes the steps in detail to isolate and expand neural stem cells in the form of neurospheres from tissue dissections of the post-natal mouse brain. Procedures for the long term passage of neurospheres and the cryopreservation of neurospheres are also provided. In addition to the guidelines and tips for generating neurosphere cultures, we describe the method to prepare neurospheres for analysis by light microscopy.

Setting Up Microdrop Cultures Protocol Protocol describes how to set up microdrop cultures to produce embryos which can then be used for making chimeras. The microdrop culture should be set up several hours to 1 day before the experiment to permit temperature and gas equilibration.

Subcloning Analysis Protocol Protocol for subcloning analysis. Includes cell lines: BG01(1), TE03, TE06, UC06(1), WA01, WA07, WA09.

Thawing Human ES Cells Protocol Protocol for thawing human ES cells.