6. Add 4 μL of 10 Â Tris-EDTA buffer and 60 μL of 2 Â proteinase K buffer.
7. Incubate the tube for 90 min at 56
C, followed by 10 min
incubation at 95
C.
8. Centrifuge the lysate at 9200 Â g for 3 min.
9. Transfer the supernatant, which contain DNA, to a new
microcentrifuge tube.
10. The extracted DNA can be used as template DNA to quantify
intracellular Leptospira by qPCR.
4 Notes
1. Instead of primary cell cultures, macrophage cell lines such as
RAW264.7 cells could be used; however, they are less
phagocytic.
2. Antibiotic susceptibility is strain dependent; thus, concentration of gentamicin should be adjusted accordingly.
3. Any species- or serovar-specific Leptospira antibody can be
used; however, an antibody raised in a different host from the
other primary antibodies should be selected in order to avoid
cross-reactivity when adding the fluorochrome-conjugated secondary antibodies.
4. Secondary antibodies that exhibit minimal cross-reaction with
heterologous host serum proteins are recommended.
5. It is important to avoid bubble formation under the coverslips
to prevent macrophage growth at the bottom of the well.
6. It is important to work with pathogenic strains that have been
in vitro cultivated for similar number of passages to obtain
comparable and reproducible results (e.g., downregulation of
many outer membrane proteins during long-term in vitro cultivation is reported).
7. Leptospira growth can be monitored by the optical density at
420 nm (OD 420 ).
8. At this step, centrifuge the volume of Leptospira culture
enough for a multiplicity of infection (MOI) of 50 (5 Â 10
7
Leptospira per well). Calculate the amount of PBS to resuspend
the bacteria after centrifugation as 100 μL per well. Prepare
two wells for each time point if you are going to perform
immunofluorescence staining and the evaluation of intracellular surviving bacteria in the same experiment.
9. Extensive washing is an essential step to decrease the background of any remaining leptospiral antigen and to ensure
204
Claudia Toma and Toshihiko Suzuki
7. Incubate the tube for 90 min at 56
C, followed by 10 min
incubation at 95
C.
8. Centrifuge the lysate at 9200 Â g for 3 min.
9. Transfer the supernatant, which contain DNA, to a new
microcentrifuge tube.
10. The extracted DNA can be used as template DNA to quantify
intracellular Leptospira by qPCR.
4 Notes
1. Instead of primary cell cultures, macrophage cell lines such as
RAW264.7 cells could be used; however, they are less
phagocytic.
2. Antibiotic susceptibility is strain dependent; thus, concentration of gentamicin should be adjusted accordingly.
3. Any species- or serovar-specific Leptospira antibody can be
used; however, an antibody raised in a different host from the
other primary antibodies should be selected in order to avoid
cross-reactivity when adding the fluorochrome-conjugated secondary antibodies.
4. Secondary antibodies that exhibit minimal cross-reaction with
heterologous host serum proteins are recommended.
5. It is important to avoid bubble formation under the coverslips
to prevent macrophage growth at the bottom of the well.
6. It is important to work with pathogenic strains that have been
in vitro cultivated for similar number of passages to obtain
comparable and reproducible results (e.g., downregulation of
many outer membrane proteins during long-term in vitro cultivation is reported).
7. Leptospira growth can be monitored by the optical density at
420 nm (OD 420 ).
8. At this step, centrifuge the volume of Leptospira culture
enough for a multiplicity of infection (MOI) of 50 (5 Â 10
7
Leptospira per well). Calculate the amount of PBS to resuspend
the bacteria after centrifugation as 100 μL per well. Prepare
two wells for each time point if you are going to perform
immunofluorescence staining and the evaluation of intracellular surviving bacteria in the same experiment.
9. Extensive washing is an essential step to decrease the background of any remaining leptospiral antigen and to ensure
204
Claudia Toma and Toshihiko Suzuki