compartments, start with the upper compartment and then
from the lower compartment; when adding medium to the
compartments, start always with the lower compartment and
then add medium to the upper compartment.
6. When you seed 500 μL of cells into 500 μL of medium in the
upper compartment of the Transwell
® plate, this will allow you
to have 2 Â 10
5 cells/cm
2 .
7. The time of incubation to achieve polarized monolayers is
dependent on cell lineage, number of cells seeded, medium
composition, and other factors. For the protocol described
here, MDCK cells took 41–43 h to achieve monolayer
establishment.
8. A high electrical resistance correlates with the presence of welldeveloped tight junctions, while a decreased in the electrical
resistance suggests a disruption in the integrity of the cell
monolayer. TEER measurements can be repeated in duplicate
or triplicate for each measurement during the experimentation.
9. The electrode should be placed into the Class II biosafety
cabinet and sterilized with UV light for 20 min before and
after use. In between measurements, the electrode can be
sterilized with 70% ethanol.
10. For the equipment EVOM2, when used for resistance measurements only, the electrode does not need to be equilibrated
or preconditioned before use.
11. Blank resistance should result in a steady ohm reading of the
solution, with an average measurement of 100 Ω/cm
2 depending on the medium. The value of the blank always adds to the
total resistance measured across a tissue culture membrane.
12. The blank resistance should be measured and then subtracted
from the resistance reading across tissue in order to obtain the
true tissue resistance. However, since the value is constant, this
procedure is not essential.
13. For the MDCK cells used, which this protocol is based, the
TEER compatible with monolayer reached a range of 200 and
300 Ω/cm
2 , which was confirmed by trypan blue assays. Those
numbers can vary accordingly to the cell lines being used, as
much as medium and other factors [17]. MDCK cells take
41–43 h to reach polarized monolayers after seeding in Transwell
® plate at a concentration of 2 Â 10
5 cells/cm
2 .
14. When using several plates in one experiment, it is ideal to
perform the trypan blue assay in one well from EACH experimental plate. However, considering that the cells, medium,
and other procedures are standardized, one well measurement,
together with the individual TEER measurement, should be
sufficient to determine monolayer establishment.
168
Elsio A. Wunder Jr.
from the lower compartment; when adding medium to the
compartments, start always with the lower compartment and
then add medium to the upper compartment.
6. When you seed 500 μL of cells into 500 μL of medium in the
upper compartment of the Transwell
® plate, this will allow you
to have 2 Â 10
5 cells/cm
2 .
7. The time of incubation to achieve polarized monolayers is
dependent on cell lineage, number of cells seeded, medium
composition, and other factors. For the protocol described
here, MDCK cells took 41–43 h to achieve monolayer
establishment.
8. A high electrical resistance correlates with the presence of welldeveloped tight junctions, while a decreased in the electrical
resistance suggests a disruption in the integrity of the cell
monolayer. TEER measurements can be repeated in duplicate
or triplicate for each measurement during the experimentation.
9. The electrode should be placed into the Class II biosafety
cabinet and sterilized with UV light for 20 min before and
after use. In between measurements, the electrode can be
sterilized with 70% ethanol.
10. For the equipment EVOM2, when used for resistance measurements only, the electrode does not need to be equilibrated
or preconditioned before use.
11. Blank resistance should result in a steady ohm reading of the
solution, with an average measurement of 100 Ω/cm
2 depending on the medium. The value of the blank always adds to the
total resistance measured across a tissue culture membrane.
12. The blank resistance should be measured and then subtracted
from the resistance reading across tissue in order to obtain the
true tissue resistance. However, since the value is constant, this
procedure is not essential.
13. For the MDCK cells used, which this protocol is based, the
TEER compatible with monolayer reached a range of 200 and
300 Ω/cm
2 , which was confirmed by trypan blue assays. Those
numbers can vary accordingly to the cell lines being used, as
much as medium and other factors [17]. MDCK cells take
41–43 h to reach polarized monolayers after seeding in Transwell
® plate at a concentration of 2 Â 10
5 cells/cm
2 .
14. When using several plates in one experiment, it is ideal to
perform the trypan blue assay in one well from EACH experimental plate. However, considering that the cells, medium,
and other procedures are standardized, one well measurement,
together with the individual TEER measurement, should be
sufficient to determine monolayer establishment.
168
Elsio A. Wunder Jr.