8. Wash the plate three times with permeabilization/blocking
buffer (about 10 min each wash).
9. Incubate the plate for 1 h at 37
C with the desired secondary
antibody conjugated to fluorochrome (e.g., TRITCconjugated donkey anti-goat).
10. Wash the plate three times with permeabilization/blocking
buffer (about 10 min each wash) (see Note 14).
11. Incubate the plate for 1 h at 37
C with anti-Leptospira
antibody.
12. Wash the plate three times with permeabilization/blocking
buffer (about 10 min each wash).
13. Incubate the plate for 1 h at 37
C with the desired secondary
antibody conjugated to fluorochrome (e.g., Cy5-conjugated
donkey anti-rabbit).
14. Wash the plate three times with permeabilization/blocking
buffer (about 10 min each wash).
15. Incubate the plate for 2 h at RT with anti-LAMP 1 (1:100 in
permeabilization/blocking buffer).
16. Wash the plate three times with permeabilization/blocking
buffer (about 10 min each wash).
17. Incubate the plate for 1 h at 37
C with the desired secondary
antibody conjugated to fluorochrome (e.g., FITC-conjugated
donkey anti-rat).
18. Wash the plate three times with permeabilization/blocking
buffer (about 10 min each wash).
19. Wash the plate twice with DW (see Note 15).
20. Mount the coverslips on a slide glass with mounting medium.
21. Analyze the samples using a confocal laser scanning microscope
(see Note 16).
3.5 Evaluation of
Intracellular Surviving
Bacteria
1. At each time point, transfer carefully each coverslip into a well
of a 6-well plate containing 1 mL of distilled water, and detach
the macrophages using a cell scrapper.
2. Forcefully pipette up and down to lyse and disaggregate the
cells.
3. To evaluate surviving bacteria by culturing, transfer 0.1 mL of
the suspended cells to 2 mL of EMJH medium, and incubate at
30
C for more than 4 days. Check daily by dark-field microscope for the presence of motile leptospires.
4. To evaluate intracellular bacteria by qPCR, transfer 0.1 mL of
the suspended cells to a microcentrifuge tube, and centrifuge at
20,000 Â g for 10 min.
5. Aspirate carefully the supernatant and resuspend the cells in
36 μL of distilled water.
Macrophage Escape Assays
203
buffer (about 10 min each wash).
9. Incubate the plate for 1 h at 37
C with the desired secondary
antibody conjugated to fluorochrome (e.g., TRITCconjugated donkey anti-goat).
10. Wash the plate three times with permeabilization/blocking
buffer (about 10 min each wash) (see Note 14).
11. Incubate the plate for 1 h at 37
C with anti-Leptospira
antibody.
12. Wash the plate three times with permeabilization/blocking
buffer (about 10 min each wash).
13. Incubate the plate for 1 h at 37
C with the desired secondary
antibody conjugated to fluorochrome (e.g., Cy5-conjugated
donkey anti-rabbit).
14. Wash the plate three times with permeabilization/blocking
buffer (about 10 min each wash).
15. Incubate the plate for 2 h at RT with anti-LAMP 1 (1:100 in
permeabilization/blocking buffer).
16. Wash the plate three times with permeabilization/blocking
buffer (about 10 min each wash).
17. Incubate the plate for 1 h at 37
C with the desired secondary
antibody conjugated to fluorochrome (e.g., FITC-conjugated
donkey anti-rat).
18. Wash the plate three times with permeabilization/blocking
buffer (about 10 min each wash).
19. Wash the plate twice with DW (see Note 15).
20. Mount the coverslips on a slide glass with mounting medium.
21. Analyze the samples using a confocal laser scanning microscope
(see Note 16).
3.5 Evaluation of
Intracellular Surviving
Bacteria
1. At each time point, transfer carefully each coverslip into a well
of a 6-well plate containing 1 mL of distilled water, and detach
the macrophages using a cell scrapper.
2. Forcefully pipette up and down to lyse and disaggregate the
cells.
3. To evaluate surviving bacteria by culturing, transfer 0.1 mL of
the suspended cells to 2 mL of EMJH medium, and incubate at
30
C for more than 4 days. Check daily by dark-field microscope for the presence of motile leptospires.
4. To evaluate intracellular bacteria by qPCR, transfer 0.1 mL of
the suspended cells to a microcentrifuge tube, and centrifuge at
20,000 Â g for 10 min.
5. Aspirate carefully the supernatant and resuspend the cells in
36 μL of distilled water.
Macrophage Escape Assays
203