3.2 Leptospira
Culture
1. Grow bacterial culture for 5–7 days at 30
C in EMHJ broth
until it reached exponential phase (~1 Â 10
8 /mL) (see Note 6).
2. The day of macrophage infection, count bacteria in a Neubauer
chamber (see Note 7).
3. Centrifuge bacteria 10,000 Â g for 10 min and resuspend in the
proper amount of PBS (see Note 8).
4. Proceed as indicated in step 2 in Subheading 3.3.
3.3 Macrophage
Infection
1. Aspirate the RPMI medium containing 10% FBS, and replace
with 4 mL of starvation and washing medium for 1 h at 37
C
in a 5% CO 2 atmosphere.
2. Add 100 μL of the Leptospira suspension to each well, and
centrifuge the plate at 500 Â g for 10 min to synchronize the
phagocytosis process.
3. Place cells at 37
C in a 5% CO 2 atmosphere.
4. At 1 h post-infection (p.i.), wash the coverslips extensively with
pre-warmed RPMI medium.
5. Transfer the coverslips to a new 6-well plate containing RPMI
medium with gentamicin (2 mL/well) to kill extracellular
bacteria.
6. After 1 h of gentamicin treatment (2 h p.i.), wash extensively
the coverslips again with pre-warmed RPMI medium to
remove remaining loosely attached bacteria.
7. Transfer the coverslip to a new 6-well plate containing RPMI
medium (see Note 9).
8. Place cells at 37
C in a 5% CO 2 atmosphere.
9. At each desire time point, proceed as indicated in step 1 in
Subheading 3.4 or step 1 in Subheading 3.5.
3.4 Immunofluorescence Staining
of Leptospira, LAMP1,
and Cathepsin D
1. At each time point, transfer carefully one coverslip into a well of
a 6-well plate, containing 2 mL of 2% PFA to fix.
2. Keep the coverslips at 4
C, overnight.
3. Transfer each coverslip into a well of a 6-well plate containing
2 mL of cold-methanol for postfixation.
4. Incubate the plate for 10 min on ice or at À20
C (see Note
10).
5. Wash the plate three times with TBS (see Note 11).
6. Aspirate TBS from the last wash, add 2 mL of permeabilization/blocking buffer, and incubate 2 h at room temperature
(RT) (see Note 12).
7. Incubate the plate for 1 h at 37
C with anti-cathepsin D
antibody (see Note 13).
202
Claudia Toma and Toshihiko Suzuki
Culture
1. Grow bacterial culture for 5–7 days at 30
C in EMHJ broth
until it reached exponential phase (~1 Â 10
8 /mL) (see Note 6).
2. The day of macrophage infection, count bacteria in a Neubauer
chamber (see Note 7).
3. Centrifuge bacteria 10,000 Â g for 10 min and resuspend in the
proper amount of PBS (see Note 8).
4. Proceed as indicated in step 2 in Subheading 3.3.
3.3 Macrophage
Infection
1. Aspirate the RPMI medium containing 10% FBS, and replace
with 4 mL of starvation and washing medium for 1 h at 37
C
in a 5% CO 2 atmosphere.
2. Add 100 μL of the Leptospira suspension to each well, and
centrifuge the plate at 500 Â g for 10 min to synchronize the
phagocytosis process.
3. Place cells at 37
C in a 5% CO 2 atmosphere.
4. At 1 h post-infection (p.i.), wash the coverslips extensively with
pre-warmed RPMI medium.
5. Transfer the coverslips to a new 6-well plate containing RPMI
medium with gentamicin (2 mL/well) to kill extracellular
bacteria.
6. After 1 h of gentamicin treatment (2 h p.i.), wash extensively
the coverslips again with pre-warmed RPMI medium to
remove remaining loosely attached bacteria.
7. Transfer the coverslip to a new 6-well plate containing RPMI
medium (see Note 9).
8. Place cells at 37
C in a 5% CO 2 atmosphere.
9. At each desire time point, proceed as indicated in step 1 in
Subheading 3.4 or step 1 in Subheading 3.5.
3.4 Immunofluorescence Staining
of Leptospira, LAMP1,
and Cathepsin D
1. At each time point, transfer carefully one coverslip into a well of
a 6-well plate, containing 2 mL of 2% PFA to fix.
2. Keep the coverslips at 4
C, overnight.
3. Transfer each coverslip into a well of a 6-well plate containing
2 mL of cold-methanol for postfixation.
4. Incubate the plate for 10 min on ice or at À20
C (see Note
10).
5. Wash the plate three times with TBS (see Note 11).
6. Aspirate TBS from the last wash, add 2 mL of permeabilization/blocking buffer, and incubate 2 h at room temperature
(RT) (see Note 12).
7. Incubate the plate for 1 h at 37
C with anti-cathepsin D
antibody (see Note 13).
202
Claudia Toma and Toshihiko Suzuki