PGC isolation was carried out as previously described4 (link). Briefly, the embryonic trunk (E10.5) or genital ridge (E11.5-E14.5) was digested at 37°C for 3 min using 0.05% Trypsin-EDTA (1x) (Gibco) or TrypLE Express (Thermo). Enzymatic digestion was followed by neutralization with DMEM/F-12 (Gibco) containing 15% foetal bovine serum (Gibco) and manual dissociation by pipetting. Following centrifugation, cells were re-suspended in DMEM/F-12 supplemented with hyaluronidase (300 µg/ml; Sigma), and a single cell suspension was generated by manual pipetting. Following centrifugation, cells were re-suspended in ice-cold PBS supplemented with poly-vinyl alcohol (10 µg/ml) and EGTA (0.4 mg/ml, Sigma). GFP positive cells were isolated using an Aria IIu (BD Bioscience) or Aria III (BD Bioscience) flow cytometer and sorted into ice cold PBS supplemented with poly-vinyl alcohol (10 µg/ml) and EGTA (0.4 mg/ml, Sigma).
Protocol detail
Isolation of Embryonic Germ Cell Progenitors
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Aria
Cells
Centrifugation
Cold
Digestion
Edta
Egta
Embryonic
Enzymatic
Foetal bovine serum
Genital
Hyaluronidase
Isolation
Poly vinyl alcohol
Trypsin
Corresponding Organization :
Other organizations : MRC London Institute of Medical Sciences, New England Biolabs (United States), Imperial College London
Top 5 similar protocols
Variable analysis
independent variables
- Enzymatic digestion method (0.05% Trypsin-EDTA or TrypLE Express)
- Developmental time point (E10.5 embryonic trunk or E11.5-E14.5 genital ridge)
- Isolation of GFP positive cells using flow cytometry
- Incubation time for enzymatic digestion (3 min)
- Supplementation of DMEM/F-12 medium with 15% fetal bovine serum
- Use of hyaluronidase (300 µg/ml) to generate single cell suspension
- Resuspension of cells in ice-cold PBS supplemented with poly-vinyl alcohol (10 µg/ml) and EGTA (0.4 mg/ml)
- Sorting of GFP positive cells into ice-cold PBS supplemented with poly-vinyl alcohol (10 µg/ml) and EGTA (0.4 mg/ml)
- No positive or negative controls were explicitly mentioned in the provided information.
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!