Nov 17, 2023

Public workspaceIsolation of intestinal organoids from matrigel for protein or RNA extraction

DOI
https://dx.doi.org/10.17504/protocols.io.36wgq37kolk5/v1
  • rachel.bates 1
  • 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
ReagentCultrex® 3-D Culture Matrix™ Reduced Growth Factor Basement Membrane Extract, PathClear®Merck MilliporeSigma (Sigma-Aldrich)Catalog #3445-001-01
ReagentPhosphate Buffered SalineThermo Fisher ScientificCatalog #28374
ReagentCell Recovery solutionCorningCatalog #354253
Intestinal organoids are grown in Amount30 µL ReagentCultrex® 3-D Culture Matrix™ Reduced Growth Factor Basement Membrane Extract, PathClear®Merck MilliporeSigma (Sigma-Aldrich)Catalog #3445-001-01 domes for Duration240:00:00

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

AddReagentCell Recovery solutionCorningCatalog #354253 Amount1 mL per well. incubate on ice for Duration01:00:00 with orbital shaking at Shaker100 rpm, 4°C .

1h
Harvest well contents into centrifuge tube. Centrifuge Centrifigation6000 rpm, 00:01:00 . Carefully remove supernatant and add Amount1 mL of Temperature4 °C PBS. Incubate on ice for Duration00:05:00 .

6m
Centrifuge Centrifigation6000 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