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INTESTINAL Publication-derived

Intestinal Organoids for Infection and Regeneration

Full SOP with Academic or a plan

Source Nigro et al., 2014 · Institut Pasteur, Paris · 10.1016/j.chom.2014.05.003

Authors: Giulia Nigro, Raffaella Rossi, Pierre-Henri Commere, Philippe Jay, Philippe J. Sansonetti Duration: 72 days 10 phases Cell source: Mouse Lgr5+ intestinal stem cells

Abstract

This protocol uses mouse intestinal crypt organoids to study how the bacterial peptidoglycan derivative muramyldipeptide (MDP) protects intestinal stem cells through Nod2 signaling, independent of epithelial proliferation. The method combines in vitro organoid culture with in vivo doxorubicin-induced stress to demonstrate that microbiota-derived molecules promote epithelial regeneration through direct stem cell cytoprotection.

Cell source
Mouse Lgr5+ intestinal stem cells
Application
Disease modeling and stem cell protection assay

Protocol overview

43 steps across 10 phases

Phase 1 · Microbiota depletion and MDP supplementation Days 1-18
  1. a Antibiotic treatment via drinking water
  2. b MDP administration initiation
Phase 2 · Intestinal crypt isolation and organoid embedding Days 0-1
  1. a Small intestine isolation and preparation
  2. b Crypt release in EDTA solution
  3. c MAMP stimulation of crypts
  4. d Crypt embedding in matrigel
  5. e Addition of culture medium
Phase 3 · Organoid culture and monitoring Days 2-10+
  1. a Daily organoid monitoring
  2. b Medium exchange every 4 days
  3. c Organoid counting and size measurement
  4. d Long-term culture for organoid development
Phase 4 · EdU proliferation assay and immunofluorescence Day 4 or endpoint
  1. a EdU pulse labeling for flow cytometry
  2. b Organoid dissociation and sample preparation
  3. c Click-iT EdU detection and fixation
  4. d Flow cytometry analysis
  5. e Ki67 immunofluorescence staining
Phase 5 · Doxorubicin in vivo stress and tissue analysis Days 1-72 (post-injection)
  1. a Doxorubicin intraperitoneal injection
  2. b Tissue harvest at defined timepoints
  3. c Tissue fixation and processing
  4. d Caspase-3 immunostaining for apoptosis detection
  5. e Image acquisition and crypt analysis
Phase 6 · EdU proliferation index and regenerative crypt assessment in vivo Post-harvest tissue sections
  1. a EdU in vivo administration
  2. b Click-iT EdU imaging on paraffin sections
  3. c Proliferation Index (PI) calculation
Phase 7 · Single cell sorting, RNA extraction, and RT-PCR Day 0-1
  1. a Crypt dissociation to single cells
  2. b Cell washing and antibody staining
  3. c Cell resuspension and filtering
  4. d Flow cytometry sorting
  5. e RNA extraction
  6. f Reverse transcription
  7. g Real-time PCR (qPCR)
Phase 8 · Single stem and Paneth cell coculture experiments Days 0-10
  1. a Cell resuspension and plating
  2. b Centrifugation and matrigel addition
  3. c Culture and MAMP stimulation
  4. d Organoid enumeration
Phase 9 · Confocal microscopy and immunofluorescence of tissue sections Post-harvest
  1. a Tissue preparation for vibratome sectioning
  2. b Antibody staining for Lysozyme and Ki67
  3. c Confocal laser-scanning microscopy
  4. d Image analysis and localization
Phase 10 · In vitro doxorubicin/H₂O₂ stress organoid assay Days 0-4
  1. a Crypt isolation and embedding
  2. b Stress induction
  3. c MDP stimulation of stressed crypts
  4. d Organoid recovery and counting

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Attribution

This SOP was written by Organthis based on the published method in Nigro et al., 2014. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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