Dear, I have problems in the first steps: EBs do not form and the cells remain single. Does anyone have the same difficulty?

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chiara fasano
Sep 13, 2023
protocol Protocol: Generation of Human iPSC-derived Neural Progenitor Cells (NPCs) as Drug Discovery Model for Neurological and Mitochondrial Disorders

I used 0.5 ml/well of accutase for 3 minutes at 37C. Then I added 4 ml/well of PBS and I did the optional centrifuge at 300g 3 minutes (the minimum g of the centrifuge in my lab). In the end I resuspended cells in 5 ml of M1.

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Alessandro Prigione Author Answered Sep 19, 2023

Heinrich Heine University Düsseldorf

Dear Chiara

Sometime the success of EBs could dipend by the quality of your PSC culture. You could also try alternative methods for generating EBs, like the hanging drop method. For example here you can find a review describing various EBs methods: https://pubmed.ncbi.nlm.nih.gov/25900308/.

Kind regards,

Alessandro

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Annika Zink Author Answered Sep 18, 2023

Heinrich Heine University Düsseldorf

Dear Chiara Fasano,


you can try and add 10 µM Rock-Inhibitor (Y-27632) to the M1 media for the first 24hours. We have observed the same problem for some iPSC-lines. The addition of 10 µM Rock Inhibitor usually helps.


Best,

Annika


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chiara fasano Answered Sep 18, 2023

IRCCS Istituto Neurologico Carlo Besta

Dear Annika Zink,


thanks for the suggestion, I will try to add Rock Inhibitor.


Best,

Chiara

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