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

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

Bio-protocol (2017-06-03)
Beth Mann, Lip Nam Loh, Geli Gao, Elaine Tuomanen
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Lithium chloride, for molecular biology, ≥99%
Sigma-Aldrich
Ethylenediaminetetraacetic acid disodium salt dihydrate, suitable for electrophoresis, for molecular biology, 99.0-101.0% (titration)
Sigma-Aldrich
Nicotinic acid, ≥98%
Sigma-Aldrich
(−)-Riboflavin, from Eremothecium ashbyii, ≥98%
Sigma-Aldrich
Hydrochloric acid, ACS reagent, 37%
Sigma-Aldrich
Potassium phosphate monobasic, powder, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.0%
Sigma-Aldrich
L-Asparagine monohydrate, suitable for cell culture, BioReagent
Sigma-Aldrich
L-Glutamine, meets USP testing specifications, suitable for cell culture, 99.0-101.0%, from non-animal source
Sigma-Aldrich
Potassium phosphate dibasic, ACS reagent, ≥98%