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INTESTINAL Publication-derived

The Cytosolic Bacterial Peptidoglycan Sensor Nod2 Affords Stem Cell Protection and Links Microbes to Gut Epithelial Regeneration

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

👤 Giulia Nigro, Raffaella Rossi, Pierre-Henri Commere, Philippe Jay, Philippe J. Sansonetti ⏱ 72 days 📋 10 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Nigro et al., 2014. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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