Hi, I came across this protocol and had a couple of questions...

AS
Anu Sebin
Jul 12, 2021
protocol Protocol: Fluorescence-based Single-cell Analysis of Whole-mount-stained and Cleared Microtissues and Organoids for High Throughput Screening
Hi,
I came across this protocol and had a couple of questions - Does each well has only one organoid? If yes, how can one make sure, the organoid is not lost during so many wash steps?

Thanks!
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Henrik Renner Author Answered Jul 14, 2021

Max Planck Institute for Molecular Biomedicine Münster

Dear Anu Sebin,

Thank you very much for your question and interest in our protocol. Using our standard workflow for the generation of organoids each well does, indeed, contain exactly one organoid. Please also refer to our previous publications (https://elifesciences.org/articles/52904 and https://bio-protocol.org/e4049) for more information on organoid generation and culture. If your culture system generates several organoids per well, this should also be compatible with the procedure we outlined here. However, we have not optimized this approach.

In order to prevent the loss of samples during the various washing steps, careful liquid handling is crucial. To facilitate the aspiration of liquids, we recommend slightly tilting the plate and performing all liquid handling by pipetting (not by vacuum pumps or similar devices). Leave a small amount of liquid (≤ 10 μl) in the well so as to not damage the organoids by extensive direct contact with a pipet tip.

Best regards,
Henrik Renner & Jan Bruder
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