16. Add 500 μL of the medium containing 4 Â 10
5 cells/mL to the
upper compartment of each well on the plate (see Note 6).
17. Incubate at 37
C with 5% CO 2 (see Note 7).
18. Wash cells daily with PBS, adding fresh medium to the compartments (see Note 5).
3.3 Measuring Cell
Monolayer Resistance
1. Evaluate the transepithelial/transendothelial electrical resistance (TEER) daily to see if the monolayer is continuous (see
Note 8).
2. Sterilize the electrode (see Note 9).
3. Connect the electrode to the meter.
4. Precondition the electrode, by placing it in your culture
medium for a few minutes (see Note 10).
5. Set the Function switch to Ohms.
6. Measure the blank resistance by placing the electrode in a cup
or a cell culture insert filled with the growth medium without
cells. Record the value (see Note 11).
7. Perform your experimental resistance measurements, cleaning
the electrode between each reading (see Note 9).
8. To obtain the true resistance value of your cultured cell monolayers, subtract the blank resistance value (see Note 12).
9. When you finish making measurements, clean and store your
electrodes dry.
3.4 Confirmation of
Polarized Cell
Monolayer
1. When cells reach the electrical resistance considered to be
compatible with established polarized cell monolayers, an
extra assay can be performed to confirm this status (see Note
13).
2. Empty the compartments from one well and wash the cells
twice with PBS (see Note 14).
3. Add 1.5 mL of PBS to the lower compartment.
4. Add 500 μL of the trypan blue solution to the upper
compartment.
5. Wait for 5 min, and visually check to see if any trypan blue has
seeped into the lower compartment (see Note 15).
6. If no trypan blue goes through, the monolayer is established,
and the cells are considered to be ready for the infection
experiment.
7. If the trypan blue goes through, the cells are not in an established monolayer and should be kept in culture until the
expected results are achieved.
Cell Monolayer Translocation Assay
165
5 cells/mL to the
upper compartment of each well on the plate (see Note 6).
17. Incubate at 37
C with 5% CO 2 (see Note 7).
18. Wash cells daily with PBS, adding fresh medium to the compartments (see Note 5).
3.3 Measuring Cell
Monolayer Resistance
1. Evaluate the transepithelial/transendothelial electrical resistance (TEER) daily to see if the monolayer is continuous (see
Note 8).
2. Sterilize the electrode (see Note 9).
3. Connect the electrode to the meter.
4. Precondition the electrode, by placing it in your culture
medium for a few minutes (see Note 10).
5. Set the Function switch to Ohms.
6. Measure the blank resistance by placing the electrode in a cup
or a cell culture insert filled with the growth medium without
cells. Record the value (see Note 11).
7. Perform your experimental resistance measurements, cleaning
the electrode between each reading (see Note 9).
8. To obtain the true resistance value of your cultured cell monolayers, subtract the blank resistance value (see Note 12).
9. When you finish making measurements, clean and store your
electrodes dry.
3.4 Confirmation of
Polarized Cell
Monolayer
1. When cells reach the electrical resistance considered to be
compatible with established polarized cell monolayers, an
extra assay can be performed to confirm this status (see Note
13).
2. Empty the compartments from one well and wash the cells
twice with PBS (see Note 14).
3. Add 1.5 mL of PBS to the lower compartment.
4. Add 500 μL of the trypan blue solution to the upper
compartment.
5. Wait for 5 min, and visually check to see if any trypan blue has
seeped into the lower compartment (see Note 15).
6. If no trypan blue goes through, the monolayer is established,
and the cells are considered to be ready for the infection
experiment.
7. If the trypan blue goes through, the cells are not in an established monolayer and should be kept in culture until the
expected results are achieved.
Cell Monolayer Translocation Assay
165