Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

3-LGS culture

EO Elizabeth Oliver
JA João Pedro Alves-Lopes
FH Femke Harteveld
RM Rod T. Mitchell
EÅ Elisabet Åkesson
OS Olle Söder
JS Jan-Bernd Stukenborg
request Request a Protocol
ask Ask a question
Favorite

Dissociated embryonic gonad tissue was cultured in a 3-LGS as previously described [34]. Briefly, dissociated cells and small aggregates were suspended in Matrigel (Corning, 356231) diluted 1:1 in MEM-α, 1% (vol/vol) Penicillin/Streptomycin at a concentration of 44, 000, 000 cells per ml (132, 000 cells in 3 μl). Three concentric drops of Matrigel (5 μl Matrigel, 3 μl Matrigel-cell suspension, 8 μl Matrigel) were sequentially plated onto the underside cell membrane of a hanging cell insert and allowed to solidify. Hanging cell inserts were positioned in 24-well plates (VWR, 734-2325) and suspended in 600 μl of NutriStem supplemented with 10% KnockOut serum replacement (Gibco, 10828-028) and 1% Penicillin/Streptomycin. Organoids were maintained under humidified conditions at 37 °C (5% CO2) for up to 14 days and 300 μl medium was replaced every second day.

Copyright and License information: The Author(s) ©2021
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A