Pathogenic L. interrogans can adhere very strongly to biotic
and abiotic surfaces; thus, it is crucial to extensively wash extracellular leptospires to accurately evaluate the fate of intracellular bacteria. In this chapter, we describe a protocol of macrophage
infection, in which extracellular bacteria are completely removed
in order to study the intracellular trafficking of Leptospira by fluorescent labeling bacteria and different markers of the phagocytic
pathway. We also describe a method to evaluate the bacterial survival within macrophages.
2 Materials
2.1 Seeding
Macrophages on
Culture Plates
1. Bone marrow-derived macrophages (BMDMs) (see Note 1).
2. Sterile coverslips (18 Â 24 cm).
3. Tissue culture 6-well plates.
4. Macrophage seeding medium: RPMI medium supplemented
with 10% fetal bovine serum (FBS).
5. Conical sterile polypropylene centrifuge tubes (50 mL).
6. Cell counting chamber.
2.2 Leptospira
Culture
1. Ellinghausen–McCullough–Johnson–Harris (EMJH) broth:
Difco Leptospira Medium Base EMJH (Becton Dickinson),
Difco Leptospira Enrichment EMJH (Becton Dickinson),
100 μg/mL of 5-fluorouracil. For 1 L of EMJH liquid
medium, dissolve 2.3 g of Leptospira Medium Base EMJH
into 900 mL distilled water, and sterilize by autoclaving. Add
100 mL of Leptospira Enrichment EMJH and 10 mL of
0.22 μm-filtrated 10 mg/mL 5-fluorouracil, and then aliquot
as needed for experiments.
2. Dark-field microscope.
3. Polypropylene microcentrifuge tubes (1.5 mL).
4. Phosphate-buffered saline (PBS): 2.68 mM KCl, 1.47 mM
KH 2 PO 4 , 136.89 mM NaCl, 8.10 mM Na 2 HPO 4 , pH 7.4.
5. Cell counting chamber.
2.3 Macrophage
Infection
1. Starvation and washing medium: RPMI without FBS.
2. Plate centrifuge.
3. Extracellular Leptospira-killing medium: RPMI with 25 μg/mL
gentamicin (see Note 2).
4. Tissue culture 6-well plates.
200
Claudia Toma and Toshihiko Suzuki
and abiotic surfaces; thus, it is crucial to extensively wash extracellular leptospires to accurately evaluate the fate of intracellular bacteria. In this chapter, we describe a protocol of macrophage
infection, in which extracellular bacteria are completely removed
in order to study the intracellular trafficking of Leptospira by fluorescent labeling bacteria and different markers of the phagocytic
pathway. We also describe a method to evaluate the bacterial survival within macrophages.
2 Materials
2.1 Seeding
Macrophages on
Culture Plates
1. Bone marrow-derived macrophages (BMDMs) (see Note 1).
2. Sterile coverslips (18 Â 24 cm).
3. Tissue culture 6-well plates.
4. Macrophage seeding medium: RPMI medium supplemented
with 10% fetal bovine serum (FBS).
5. Conical sterile polypropylene centrifuge tubes (50 mL).
6. Cell counting chamber.
2.2 Leptospira
Culture
1. Ellinghausen–McCullough–Johnson–Harris (EMJH) broth:
Difco Leptospira Medium Base EMJH (Becton Dickinson),
Difco Leptospira Enrichment EMJH (Becton Dickinson),
100 μg/mL of 5-fluorouracil. For 1 L of EMJH liquid
medium, dissolve 2.3 g of Leptospira Medium Base EMJH
into 900 mL distilled water, and sterilize by autoclaving. Add
100 mL of Leptospira Enrichment EMJH and 10 mL of
0.22 μm-filtrated 10 mg/mL 5-fluorouracil, and then aliquot
as needed for experiments.
2. Dark-field microscope.
3. Polypropylene microcentrifuge tubes (1.5 mL).
4. Phosphate-buffered saline (PBS): 2.68 mM KCl, 1.47 mM
KH 2 PO 4 , 136.89 mM NaCl, 8.10 mM Na 2 HPO 4 , pH 7.4.
5. Cell counting chamber.
2.3 Macrophage
Infection
1. Starvation and washing medium: RPMI without FBS.
2. Plate centrifuge.
3. Extracellular Leptospira-killing medium: RPMI with 25 μg/mL
gentamicin (see Note 2).
4. Tissue culture 6-well plates.
200
Claudia Toma and Toshihiko Suzuki