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. 2016 Dec 5;6(23):e2037.
doi: 10.21769/BioProtoc.2037.

Preparation of Purified Gram-positive Bacterial Cell Wall and Detection in Placenta and Fetal Tissues

Beth Mann  1 , Lip Nam Loh  1 , Geli Gao  1 , Elaine Tuomanen  1
Affiliations

Preparation of Purified Gram-positive Bacterial Cell Wall and Detection in Placenta and Fetal Tissues

Beth Mann et al. Bio Protoc. .

Abstract

Cell wall is a complex biopolymer on the surface of all Gram-positive bacteria. During infection, cell wall is recognized by the innate immune receptor Toll-like receptor 2 causing intense inflammation and tissue damage. In animal models, cell wall traffics from the blood stream to many organs in the body, including brain, heart, placenta and fetus. This protocol describes how to prepare purified cell wall from Streptococcus pneumoniae, detect its distribution in animal tissues, and study the tissue response using the placenta and fetal brain as examples.

Keywords: Bacterial inflammation; Cell wall; Fetal neurogenesis; Neuroproliferation; Peptidoglycan; Placental trafficking; Streptococcus pneumonae; Toll like receptor 2 ligand.

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Figures

Figure 1
Figure 1. Overnight growth of unenapsulated strain R6 on TSA blood agar
Pure culture of pneumococcus is the source of bacteria for seed stocks made in step A1b.
Figure 2
Figure 2. Final appearance of purified pneumococcal CW
Figure 3
Figure 3. Final appearance of FITC labeled pneumococcal CW
Figure 4
Figure 4. Cross sections of mouse brain
For sectioning, make incision along dotted line. Coronal section is preferred to remove cerebellum and create flat surface for embedding the cortex.
Figure 5
Figure 5. FITC CW in the fetal brain or placenta is documented and quantified by microscopy
Confocal images of CW distribution in placental and fetal brain tissue (arrows) are analyzed using ImageJ.
See this image and copyright information in PMC

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