I want to look at the influence of certain compounds on the organoids. I am looking for a high-throughput assays using plate reader. One assay I could think of is to analyze proliferation by measuring ATP production. What other assays can I do in a 96-well format and analyze on a plate reader. Also, how do I normalize for the differences in the number/size of organoids in different wells?
Would be happy to get suggestions if anyone has any experience with this. Thanks!
Hello Shilpa Patil
You can refer to the research article given below. You will find some interesting assays which could be analyzed on a plate reader.
Article A fast and simple fluorometric method to detect cell death i...
Best.
Dear Shilpa,
We have used for several years the Presto Blue kit from Thermo Fisher to measure cell viability on intestinal organoids. The viability of organoids can be evaluated across several time points on the same plate and do not require lysis (which means you can get a day 0 reference before treatment to account for organoid variability across wells). We did drug screening on 96 well plates using this method and it only requires a plate reader.
The method is described in several of our papers. See our Cell Stem Cell paper below for example.
https://www.sciencedirect.com/science/article/abs/pii/S1934590920302885
Regards,
Thierry
Thierry Jarde Hi Thierry. I have read your paper. It is very interesting. Just one question. As Shilpa Patil said, how you normalized the size of organoid? As it maybe different size of organoid in the wells. Thank you so much for sharing again.
Thierry Jarde Thanks for this. I am yet to try but looks like I can give a shot.
Ziming Chen When you seed the organoids, you make them into single cells, count and then seed same number of cells into each well. Once there cells form organoids, you can start the treatment.
Shilpa Patil Thanks so much for your information. I just afraid the organoid size would change after treatment even organoids were relatively same before treatment, especially for long-term treatment. I am wondering if possible to use protein content to normalize it, but it will be better to have some literature for support.
Hi Ziming Chen
I agree with Shilpa Patil . We would dissociate organoids into single cells, quantify cell number and reseed single cells/fragments at the desired density.
In addition, once organoids are formed, we use PrestoBlue at day 0 just before drug treatment to obtain a measurement of overall cell viability per well (which is used as the reference point and gives an indication of variability across wells). We repeat the same PrestoBlue assay at the end of the drug treatment period (at day 4 for example) and quantify adjusted cell viability per well by dividing day 4 values by day 0 values. This way, the potential plating errors are taken into consideration.
Regards,
Thierry