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INTESTINAL Publication-derived

Human Clostridium difficile Infection: Inhibition of NHE3 and Microbiota Profile

Source Engevik et al., 2015 · University of Cincinnati College of Medicine · 10.1152/ajpgi.00090.2014

👤 Melinda A. Engevik, Kristen A. Engevik, Mary Beth Yacyshyn, Jiang Wang, Daniel J. Hassett, Benjamin Darien, Bruce R. Yacyshyn, Roger T. Worrell ⏱ 7 days 📋 9 phases 🧫 Human intestinal organoids (HIOs) derived from human pluripotent stem cells (hPSC)

Abstract

This protocol documents methods for studying Clostridium difficile inhibition of the Na+/H+ exchanger 3 (NHE3) in human intestinal organoids and patient biopsy specimens. The work demonstrates that C. difficile toxin downregulates NHE3 expression, altering the intestinal microenvironment to favor pathogenic bacterial growth and microbiota dysbiosis observed in recurrent CDI patients.

Cell source
Human intestinal organoids (HIOs) derived from human pluripotent stem cells (hPSC)
Application
Disease modeling

Protocol overview

41 steps across 9 phases

Human Intestinal Organoid (HIO) Generation and Culture Days 0-7
  1. 1 HIO Differentiation from hPSC
  2. 2 HIO Growth in Matrigel
  3. 3 HIO Retrieval from Culture
Bacterial Culture Preparation Days -2 to 0
  1. 1 C. difficile Culture
  2. 2 C. butyricum Culture
  3. 3 Stool Supernatant Preparation
Microinjection of HIOs Day 0
  1. 1 Microinjection Needle Preparation
  2. 2 HIO Microinjection with Bacterial Cultures or Stool Supernatant
  3. 3 Post-Injection Organoid Incubation
RNA Extraction and qRT-PCR for NHE3 mRNA Day 1
  1. 1 HIO Homogenization and Trizol Extraction
  2. 2 RNA Isolation by Chloroform Extraction
  3. 3 Reverse Transcription
  4. 4 qRT-PCR Amplification of NHE3 mRNA
  5. 5 Data Analysis and Calculation of Fold Difference
Immunofluorescence Staining of HIOs for NHE3 Protein Day 1-3
  1. 1 HIO Fixation
  2. 2 PBS Wash and Sucrose Incubation
  3. 3 OCT Embedding and Freezing
  4. 4 Cryostat Sectioning
  5. 5 Antibody Staining and Imaging
  6. 6 Image Analysis and Semiquantification
Biopsy Specimen Histology and Immunofluorescence Sample Processing
  1. 1 Tissue Collection and Fixation
  2. 2 Paraffin Sectioning
  3. 3 Hematoxylin and Eosin Staining
  4. 4 Antigen Retrieval
  5. 5 Blocking and Primary Antibody Staining
  6. 6 Secondary Antibody Incubation and Counterstaining
  7. 7 Confocal Microscopy and Image Analysis
In Vitro Bacterial Growth in Varying Na+ and pH Conditions Days 0-2
  1. 1 Preparation of Growth Media with Varying Na+ Concentrations
  2. 2 Bacterial Inoculation and Growth
  3. 3 Optical Density Measurement
  4. 4 Colony-Forming Unit (CFU) Enumeration
  5. 5 pH Optimization Studies
Quantitative PCR Analysis of Bacterial Microbiota Sample Processing
  1. 1 Total DNA Extraction from Stool
  2. 2 Design and Validation of Bacterial Primers
  3. 3 qPCR Amplification and Quantification
  4. 4 Data Analysis and Bacterial Abundance Calculation
Ion Concentration and pH Measurement in Stool Sample Processing
  1. 1 Stool Sample Preparation
  2. 2 Sodium (Na+) and Potassium (K+) Concentration Determination
  3. 3 Chloride (Cl-) Concentration Determination
  4. 4 pH Measurement
  5. 5 Anion Gap Calculation

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Engevik et al., 2015. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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