2.4 Immunofluorescence Staining
of Leptospira, LAMP1,
and Cathepsin D
1. Tissue culture 6-well plates.
2. 2% paraformaldehyde in PBS (2% PFA).
3. Cold methanol.
4. Tris-buffered saline (TBS): 25 mM Tris–HCl, pH 7.4, 137 mM
NaCl, 2.68 mM KCl.
5. Permeabilization/blocking buffer: 0.2% saponin, 10% Blocking One (Nacalai Tesque) in TBS.
6. Goat polyclonal anti-cathepsin D (G-19, Santa Cruz
Biotechnologies).
7. Anti-Leptospira antibody (see Note 3).
8. Rat monoclonal anti-mouse LAMP1 (1D4B, eBioscience).
9. Secondary antibodies: antibodies conjugated with a fluorochrome of choice (e.g., TRITC-conjugated donkey anti-goat,
Cy5-conjugated donkey anti-rabbit, FITC-conjugated donkey
anti-rat) (see Note 4).
10. Mounting medium (e.g., Vectashield, Vector Laboratories).
11. Immunofluorescence microscope slides.
12. Confocal laser microscope.
2.5 Recovery of
Intracellular Surviving
Bacteria
1. Tissue culture 6-well plates.
2. Sterile H 2 O.
3. Cell scrappers.
4. EMJH broth.
5. 10Â Tris-EDTA buffer: 100 mM Tris-HCl, pH 8.3,
10 mM EDTA.
6. 2Â proteinase K buffer: 20 mM Tris–HCl, pH 8.3100 mM
KCl, 5 mM MgCl 2 , 1% Tween 20, 800 μg/mL of proteinase K.
7. Heat Block.
3 Methods
3.1 Seeding
Macrophages on
Culture Plates
1. Place a coverslip into each well of a 6-well plate.
2. Prepare a suspension of macrophages (around 5 Â 10
5 cells per
mL) in RPMI/10% FBS medium (e.g., for one 6-well plate,
prepare around 6 Â 10
6 cells in 12 mL).
3. Transfer 2 mL of the cell suspension (around 1 Â 10
6 cells) into
each well of a 6-well plate, and briefly shake plates to distribute
the cells evenly.
4. Push the coverslip gently with a tip to ensure that it is place at
the bottom of the well (see Note 5).
5. Incubate cells at 37
C in a 5% CO 2 atmosphere overnight to
allow cell attachment.
6. Proceed as indicated in step 1 in Subheading 3.3.
Macrophage Escape Assays
201
of Leptospira, LAMP1,
and Cathepsin D
1. Tissue culture 6-well plates.
2. 2% paraformaldehyde in PBS (2% PFA).
3. Cold methanol.
4. Tris-buffered saline (TBS): 25 mM Tris–HCl, pH 7.4, 137 mM
NaCl, 2.68 mM KCl.
5. Permeabilization/blocking buffer: 0.2% saponin, 10% Blocking One (Nacalai Tesque) in TBS.
6. Goat polyclonal anti-cathepsin D (G-19, Santa Cruz
Biotechnologies).
7. Anti-Leptospira antibody (see Note 3).
8. Rat monoclonal anti-mouse LAMP1 (1D4B, eBioscience).
9. Secondary antibodies: antibodies conjugated with a fluorochrome of choice (e.g., TRITC-conjugated donkey anti-goat,
Cy5-conjugated donkey anti-rabbit, FITC-conjugated donkey
anti-rat) (see Note 4).
10. Mounting medium (e.g., Vectashield, Vector Laboratories).
11. Immunofluorescence microscope slides.
12. Confocal laser microscope.
2.5 Recovery of
Intracellular Surviving
Bacteria
1. Tissue culture 6-well plates.
2. Sterile H 2 O.
3. Cell scrappers.
4. EMJH broth.
5. 10Â Tris-EDTA buffer: 100 mM Tris-HCl, pH 8.3,
10 mM EDTA.
6. 2Â proteinase K buffer: 20 mM Tris–HCl, pH 8.3100 mM
KCl, 5 mM MgCl 2 , 1% Tween 20, 800 μg/mL of proteinase K.
7. Heat Block.
3 Methods
3.1 Seeding
Macrophages on
Culture Plates
1. Place a coverslip into each well of a 6-well plate.
2. Prepare a suspension of macrophages (around 5 Â 10
5 cells per
mL) in RPMI/10% FBS medium (e.g., for one 6-well plate,
prepare around 6 Â 10
6 cells in 12 mL).
3. Transfer 2 mL of the cell suspension (around 1 Â 10
6 cells) into
each well of a 6-well plate, and briefly shake plates to distribute
the cells evenly.
4. Push the coverslip gently with a tip to ensure that it is place at
the bottom of the well (see Note 5).
5. Incubate cells at 37
C in a 5% CO 2 atmosphere overnight to
allow cell attachment.
6. Proceed as indicated in step 1 in Subheading 3.3.
Macrophage Escape Assays
201