3.5 Determining the
Concentration of
Eukaryotic Cell
1. Trypsinize one well of your eukaryotic cell Transwell
® plate to
check the concentration of cells (see Note 16).
2. Wash one individual well of the Transwell
® plate with PBS.
3. Add 600 μL of trypsin/EDTA solution to the lower compartment and 250 μL to the upper compartment.
4. Incubate the plate at 37
C incubator with 5% CO 2 for 10 m.
5. Scratch membrane to collect all cells into 1.5 mL tube.
6. Determine the number of eukaryotic cells with a Neubauer
chamber.
3.6 Infection of
Polarized Cell
Monolayer
1. Prepare mid-log phase Leptospira sp. cultures.
2. Leptospira biflexa serovar Patoc strain Patoc1 should be used as
a negative control for the experiment (see Note 17).
3. Leptospira interrogans serovar Copenhageni strain Fiocruz
L1-130 should be used as a positive control for the experiment
(see Note 18).
4. Perform the infection of wells in duplicate or triplicate for each
strain to be tested (see Note 19).
5. For a Leptospira strain that reaches ~10
8 cells/mL, one 5 mL
tube of culture should be enough for one Transwell
® plate.
6. Centrifuge the Leptospira sp. culture at 12,000 Â g for 10 m.
7. Remove supernatant and resuspend pelleted cells in 5 mL
of PBS.
8. Centrifuge the cells once more at 12,000 Â g for 10 m.
9. Resuspend the pellet in 200 μL of PBS.
10. Determine the number of Leptospira spp. (leptospires per milliliter) using a Petroff-Hausser counting chamber.
11. Determine the number of leptospires that need to be infected
per well using a multiplicity of infection (MOI) of 100 leptospires (1:100) (see Note 20).
12. Wash the remaining wells of the Transwell
® plate with PBS.
13. Add 1.5 mL of complete growth medium into the lower compartment of each experimental well.
14. Add 500 μL of leptospires diluted in EMJH/DMEM medium
into the upper compartment of the Transwell
® plate (see Note
20).
15. Immediately measure the TEER.
16. Incubate the plate at 37
C incubator with 5% CO 2 .
17. Determine the time points that will be used to assess the
translocation and TEER (see Note 21).
166
Elsio A. Wunder Jr.
Concentration of
Eukaryotic Cell
1. Trypsinize one well of your eukaryotic cell Transwell
® plate to
check the concentration of cells (see Note 16).
2. Wash one individual well of the Transwell
® plate with PBS.
3. Add 600 μL of trypsin/EDTA solution to the lower compartment and 250 μL to the upper compartment.
4. Incubate the plate at 37
C incubator with 5% CO 2 for 10 m.
5. Scratch membrane to collect all cells into 1.5 mL tube.
6. Determine the number of eukaryotic cells with a Neubauer
chamber.
3.6 Infection of
Polarized Cell
Monolayer
1. Prepare mid-log phase Leptospira sp. cultures.
2. Leptospira biflexa serovar Patoc strain Patoc1 should be used as
a negative control for the experiment (see Note 17).
3. Leptospira interrogans serovar Copenhageni strain Fiocruz
L1-130 should be used as a positive control for the experiment
(see Note 18).
4. Perform the infection of wells in duplicate or triplicate for each
strain to be tested (see Note 19).
5. For a Leptospira strain that reaches ~10
8 cells/mL, one 5 mL
tube of culture should be enough for one Transwell
® plate.
6. Centrifuge the Leptospira sp. culture at 12,000 Â g for 10 m.
7. Remove supernatant and resuspend pelleted cells in 5 mL
of PBS.
8. Centrifuge the cells once more at 12,000 Â g for 10 m.
9. Resuspend the pellet in 200 μL of PBS.
10. Determine the number of Leptospira spp. (leptospires per milliliter) using a Petroff-Hausser counting chamber.
11. Determine the number of leptospires that need to be infected
per well using a multiplicity of infection (MOI) of 100 leptospires (1:100) (see Note 20).
12. Wash the remaining wells of the Transwell
® plate with PBS.
13. Add 1.5 mL of complete growth medium into the lower compartment of each experimental well.
14. Add 500 μL of leptospires diluted in EMJH/DMEM medium
into the upper compartment of the Transwell
® plate (see Note
20).
15. Immediately measure the TEER.
16. Incubate the plate at 37
C incubator with 5% CO 2 .
17. Determine the time points that will be used to assess the
translocation and TEER (see Note 21).
166
Elsio A. Wunder Jr.