15. For more accurate results, the trypan blue in the lower compartment of the well can also be quantified by absorbance
measurements with spectrophotometer.
16. When using several plates in one experiment, the average of
eukaryotic cells per well should ideally be determined from one
well of EACH experimental plate. However, considering that
the cells, medium, and other procedures are standardized, one
well measurement should be sufficient to determine this number for the entire experiment.
17. Saprophyte species of Leptospira spp., e.g. Leptospira biflexa
serovar Patoc strain Patoc1, are not able to rapidly translocate
through polarized monolayers (REF) and are good controls to
make sure that your polarized monolayer is well established and
the translocation system is working.
18. The strain Fiocruz L1-130 has a well-known ability to translocate [3, 18] and for that reason is recommended. However, the
positive control should be any leptospiral strain in which ability
to translocate is well established and can be used to determine
the efficiency and accuracy of the assay. An extra positive control can be used to determine the accuracy of the TEER measurements, like a pathogenic Salmonella sp. strain [3].
19. Duplicate or triplicate infection for each strain allows for evaluation of reproducibility and variations within the experiment.
Negative and positive controls should also be tested in duplicate or triplicate. Furthermore, each plate should have the
following: one well without any cells for measurement of the
blank resistance (see Note 12), one well for the trypan blue
assay, and one well for determination of eukaryotic cell average.
20. The number of leptospires that need to be inoculated with the
eukaryotic cells is calculated based on the average number of
eukaryotic cells per well and the number of leptospires per
milliliter. The final number of leptospires should be diluted in
500 μL of the EMJH/DMEM medium.
21. Previous experiments showed that pathogenic Leptospira spp.
are able to translocate after 15 min [3]. When establishing the
times for assessment, remember to take in consideration the
amount of time that takes to measure the TEER of all the wells
and to count all the leptospires that were able to translocate in
each well. Time assessment over 8–12 h should take in consideration the generation time for leptospires.
22. Another method to calculate the ability of leptospires to translocate MDCK polarized monolayers is by calculating the proportion of leptospires measured in the lower compartment in a
well with eukaryotic monolayer in comparison to the number
of leptospires that are present in the lower compartment of a
well without eukaryotic cells.
Cell Monolayer Translocation Assay
169
measurements with spectrophotometer.
16. When using several plates in one experiment, the average of
eukaryotic cells per well should ideally be determined from one
well of EACH experimental plate. However, considering that
the cells, medium, and other procedures are standardized, one
well measurement should be sufficient to determine this number for the entire experiment.
17. Saprophyte species of Leptospira spp., e.g. Leptospira biflexa
serovar Patoc strain Patoc1, are not able to rapidly translocate
through polarized monolayers (REF) and are good controls to
make sure that your polarized monolayer is well established and
the translocation system is working.
18. The strain Fiocruz L1-130 has a well-known ability to translocate [3, 18] and for that reason is recommended. However, the
positive control should be any leptospiral strain in which ability
to translocate is well established and can be used to determine
the efficiency and accuracy of the assay. An extra positive control can be used to determine the accuracy of the TEER measurements, like a pathogenic Salmonella sp. strain [3].
19. Duplicate or triplicate infection for each strain allows for evaluation of reproducibility and variations within the experiment.
Negative and positive controls should also be tested in duplicate or triplicate. Furthermore, each plate should have the
following: one well without any cells for measurement of the
blank resistance (see Note 12), one well for the trypan blue
assay, and one well for determination of eukaryotic cell average.
20. The number of leptospires that need to be inoculated with the
eukaryotic cells is calculated based on the average number of
eukaryotic cells per well and the number of leptospires per
milliliter. The final number of leptospires should be diluted in
500 μL of the EMJH/DMEM medium.
21. Previous experiments showed that pathogenic Leptospira spp.
are able to translocate after 15 min [3]. When establishing the
times for assessment, remember to take in consideration the
amount of time that takes to measure the TEER of all the wells
and to count all the leptospires that were able to translocate in
each well. Time assessment over 8–12 h should take in consideration the generation time for leptospires.
22. Another method to calculate the ability of leptospires to translocate MDCK polarized monolayers is by calculating the proportion of leptospires measured in the lower compartment in a
well with eukaryotic monolayer in comparison to the number
of leptospires that are present in the lower compartment of a
well without eukaryotic cells.
Cell Monolayer Translocation Assay
169