Nov 17, 2023

Isolation of intestinal organoids from matrigel for protein or RNA extraction

DOI
https://dx.doi.org/10.17504/protocols.io.36wgq37kolk5/v1
  • 1UCL
rachel.bates Bates
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DOI: https://dx.doi.org/10.17504/protocols.io.36wgq37kolk5/v1
Protocol Citation: rachel.bates 2023. Isolation of intestinal organoids from matrigel for protein or RNA extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq37kolk5/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 30, 2023
Last Modified: May 31, 2024
Protocol  Integer ID: 85690
Keywords: ASAPCRN, rna extraction isolation of intestinal organoid, isolation of intestinal organoid, intestinal organoid, matrigel for protein, organoid, rna extraction isolation, matrigel, rna isolation, extraction, cultrex growth
Funders Acknowledgements:
asap
Abstract
Isolation of intestinal organoids from matrigel or cultrex growth domes ready for protein or RNA isolation.
Protocol materials
Cultrex® 3-D Culture Matrix™ Reduced Growth Factor Basement Membrane Extract, PathClear®Merck MilliporeSigma (Sigma-Aldrich)Catalog #3445-001-01
Phosphate Buffered SalineThermo Fisher ScientificCatalog #28374
Cell Recovery solutionCorningCatalog #354253
Intestinal organoids are grown in 30 µL Cultrex® 3-D Culture Matrix™ Reduced Growth Factor Basement Membrane Extract, PathClear®Merck MilliporeSigma (Sigma-Aldrich)Catalog #3445-001-01 domes for 240:00:00

1w 3d
Once organoids are large enough for harvest domes are washed three times with ice cold Phosphate Buffered SalineThermo Fisher ScientificCatalog #28374 .

AddCell Recovery solutionCorningCatalog #354253 1 mL per well. incubate on ice for 01:00:00 with orbital shaking at 100 rpm, 4°C .

1h
Harvest well contents into centrifuge tube. Centrifuge 6000 rpm, 00:01:00 . Carefully remove supernatant and add 1 mL of 4 °C PBS. Incubate on ice for 00:05:00 .

6m
Centrifuge 6000 rpm, 4°C, 00:01:00 . Carefully remove the supernatant and as much solidified matrigel as possible leaving behind organoids. Cell pellet is ready for RNA or protein extraction.

1m