3.7 Determining
Translocation Through
Polarized Cell
Monolayer
1. At each time point selected for your analysis after the period of
interaction/infection, measure the TEER and collect an aliquot of 50 μL from the bottom compartment of each well.
2. Immediately after the collection of the aliquot, proceed to
assess how many leptospires have translocated through the
cell monolayer using a Petroff-Hausser counting chamber.
3. Count in triplicate the number of leptospires that translocate in
each well, and calculate the mean and standard deviation.
4. The ability of leptospires to translocate MDCK polarized
monolayers is determined by calculating the proportion of
leptospires in the lower compartment in comparison to the
initial inoculums (see Note 22).
5. Calculate the mean and standard deviation of the percentage of
translocation of the monolayer for each leptospiral strain
tested, considering the duplicate or triplicate wells (see Note
19).
6. Plot the results in a graph, showing the percentage of translocation in a time line series, including the variation of the
average measured TEER of the wells for each strain.
4 Notes
1. The procedure described here reflects the use of Madin-Darby
Canine Kidney (MDCK) cells, and DMEM medium is ideal for
those cells. Other eukaryotic cell lines can be used for this
experiment, and the media should reflect the growth needs of
the cell line in use.
2. We used the EVOM2 epithelial voltohmmeter (World Precision Instruments, Sarasota, FL) in our experiments, and the
instruction manual can be found at https://www.wpiinc.com/
clientuploads/pdf/EVOM2_IM.pdf.
3. Cell passage number can affect the electrical resistance of
eukaryotic cell lines. Thus, it is ideal to keep a working seed
of your cells and a uniform number of passages (<30) when
performing your experiments.
4. MDCK cells are counted in a Neubauer counting chamber.
Trypan blue is used to selectively stain dead cells, as the dye is
only able to cross the membrane of a dead cell. By counting live
cells (clear) and dead cells (blue), you can determine the percentage viability of your cells, taking that in consideration when
determining the number of cells.
5. To avoid disturbing flow between the compartments, which
will perturb the cell monolayers, it is very important to always
follow this sequence: when taking out medium from the
Cell Monolayer Translocation Assay
167
Translocation Through
Polarized Cell
Monolayer
1. At each time point selected for your analysis after the period of
interaction/infection, measure the TEER and collect an aliquot of 50 μL from the bottom compartment of each well.
2. Immediately after the collection of the aliquot, proceed to
assess how many leptospires have translocated through the
cell monolayer using a Petroff-Hausser counting chamber.
3. Count in triplicate the number of leptospires that translocate in
each well, and calculate the mean and standard deviation.
4. The ability of leptospires to translocate MDCK polarized
monolayers is determined by calculating the proportion of
leptospires in the lower compartment in comparison to the
initial inoculums (see Note 22).
5. Calculate the mean and standard deviation of the percentage of
translocation of the monolayer for each leptospiral strain
tested, considering the duplicate or triplicate wells (see Note
19).
6. Plot the results in a graph, showing the percentage of translocation in a time line series, including the variation of the
average measured TEER of the wells for each strain.
4 Notes
1. The procedure described here reflects the use of Madin-Darby
Canine Kidney (MDCK) cells, and DMEM medium is ideal for
those cells. Other eukaryotic cell lines can be used for this
experiment, and the media should reflect the growth needs of
the cell line in use.
2. We used the EVOM2 epithelial voltohmmeter (World Precision Instruments, Sarasota, FL) in our experiments, and the
instruction manual can be found at https://www.wpiinc.com/
clientuploads/pdf/EVOM2_IM.pdf.
3. Cell passage number can affect the electrical resistance of
eukaryotic cell lines. Thus, it is ideal to keep a working seed
of your cells and a uniform number of passages (<30) when
performing your experiments.
4. MDCK cells are counted in a Neubauer counting chamber.
Trypan blue is used to selectively stain dead cells, as the dye is
only able to cross the membrane of a dead cell. By counting live
cells (clear) and dead cells (blue), you can determine the percentage viability of your cells, taking that in consideration when
determining the number of cells.
5. To avoid disturbing flow between the compartments, which
will perturb the cell monolayers, it is very important to always
follow this sequence: when taking out medium from the
Cell Monolayer Translocation Assay
167