establishment of polarized monolayer. Pathogen is added to the
upper compartment, and the ability of translocation is measured by
assessing the number of pathogens present in the lower compartment after determined periods of time post-infection. The integrity
of the polarized monolayers is monitored using an epithelial voltohmmeter apparatus (Fig. 1).
Here we describe an in vitro cell monolayer translocation assay
that enables the investigation of a key feature of Leptospira pathogenesis, providing assessment of the phenotype of new isolates or
potential mutants of interest. In vitro surrogates to investigate key
steps of pathogenesis contribute to the principle of replacing,
reducing, and refining the use of animals in experimentation.
2 Materials
Use sterile ultrapure water to prepare media and solutions. All
media and solutions should be pre-warmed at 37
C. Use 10%
NaOH or 1 N HCl solutions to adjust pH.
2.1 Growth and
Seeding of Eukaryotic
Cells (MDCK)
1. Complete growth medium: Dulbecco’s Modified Eagle
Medium (DMEM) + 10% fetal bovine sera (FBS) (see Note 1).
2. Phosphate buffered saline (PBS): 137 mM NaCl, 2.68 mM
KCl, 10.1 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 , pH 7.4.
3. Trypsin/EDTA solution: PBS, 0.05% trypsin, 0.02% EDTA.
4. Neubauer counting chamber.
5. 12 mm diameter Transwell
® filter units with 3 μm pores.
Fig. 1 Schematic representation showing the polarized eukaryotic cell monolayer and the components of the
system to assess leptospiral translocation, including the epithelia voltohmmeter used to measure the
epithelial/endothelial electrical resistance (TEER)
162
Elsio A. Wunder Jr.
upper compartment, and the ability of translocation is measured by
assessing the number of pathogens present in the lower compartment after determined periods of time post-infection. The integrity
of the polarized monolayers is monitored using an epithelial voltohmmeter apparatus (Fig. 1).
Here we describe an in vitro cell monolayer translocation assay
that enables the investigation of a key feature of Leptospira pathogenesis, providing assessment of the phenotype of new isolates or
potential mutants of interest. In vitro surrogates to investigate key
steps of pathogenesis contribute to the principle of replacing,
reducing, and refining the use of animals in experimentation.
2 Materials
Use sterile ultrapure water to prepare media and solutions. All
media and solutions should be pre-warmed at 37
C. Use 10%
NaOH or 1 N HCl solutions to adjust pH.
2.1 Growth and
Seeding of Eukaryotic
Cells (MDCK)
1. Complete growth medium: Dulbecco’s Modified Eagle
Medium (DMEM) + 10% fetal bovine sera (FBS) (see Note 1).
2. Phosphate buffered saline (PBS): 137 mM NaCl, 2.68 mM
KCl, 10.1 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 , pH 7.4.
3. Trypsin/EDTA solution: PBS, 0.05% trypsin, 0.02% EDTA.
4. Neubauer counting chamber.
5. 12 mm diameter Transwell
® filter units with 3 μm pores.
Fig. 1 Schematic representation showing the polarized eukaryotic cell monolayer and the components of the
system to assess leptospiral translocation, including the epithelia voltohmmeter used to measure the
epithelial/endothelial electrical resistance (TEER)
162
Elsio A. Wunder Jr.