6. Add 9 mL of pre-warmed uptake buffer (e.g., a tenfold dilution
to approximately 1 Â 10
7 cells/mL).
7. For infection of macrophages in a 24-well plate, calculate the
volume of medium needed for 0.5 mL per well and adjust the
concentration of the bacteria to 2 Â 10
6 cells/mL (e.g., a
fivefold dilution) in warm uptake buffer.
8. Keep the bacteria at 37
C until ready for the infection.
9. Prior to the day of infection, seed wells of 24-well plates with
macrophages in macrophage infection medium. If desired, cells
may be activated prior to infection (see Subheading 3.7). See
Subheading 3.9 for infection of macrophages in 96- and
384-well plates. See Note 14 for modified methods for infection of J774 and THP-1 cells.
10. On the day of infection, remove the medium and add 0.5 mL
of bacterial-uptake buffer suspension, prepared as described
above, to each well in a 24-well plate (see Note 15).
11. Incubate plates for 1 h at 37
C and 5% CO 2 .
12. Remove bacterial suspension and wash each well twice with
1 mL of pre-warmed wash medium.
13. Add 0.5 mL of fresh, pre-warmed macrophage infection
medium.
14. Incubate at 37
C and 5% CO 2 .
15. To maintain M. tuberculosis-infected macrophages, change the
medium with fresh, pre-warmed macrophage infection
medium every 2 days.
16. Observe macrophages under an inverted light microscope to
ascertain cell health. Survival assays can be continued through
14 days postinfection, although survival trends may become
variable at time points later than 9 days postinfection.
3.9 Infection of
Primary Bone MarrowDerived Macrophages
with M. tuberculosis in
96- and 384-Well
Plates
1. To infect macrophages with M. tuberculosis in a 96- or 384-well
plate, follow the recommended volumes and concentrations of
macrophages for a specific plate type as described in Note 11.
See Note 14 for modified methods for infection of J774 and
THP-1 cells.
2. Prepare M. tuberculosis as described in Subheading 3.8; however, after declumping and resuspending bacteria in uptake
buffer, adjust to 0.2 OD 600 with macrophage infection
medium.
3. For a 96-well plate, add 20 μL of the bacterial suspension to
each well, and for a 384-well plate, add 5 μL of the bacterial
suspension per well (do not remove medium prior to infection). The medium in the wells does not need to be changed or
washed during the infection, which can reliably proceed for
approximately 6 days (see Note 16).
Macrophage Infection Models for Mycobacterium tuberculosis
177
to approximately 1 Â 10
7 cells/mL).
7. For infection of macrophages in a 24-well plate, calculate the
volume of medium needed for 0.5 mL per well and adjust the
concentration of the bacteria to 2 Â 10
6 cells/mL (e.g., a
fivefold dilution) in warm uptake buffer.
8. Keep the bacteria at 37
C until ready for the infection.
9. Prior to the day of infection, seed wells of 24-well plates with
macrophages in macrophage infection medium. If desired, cells
may be activated prior to infection (see Subheading 3.7). See
Subheading 3.9 for infection of macrophages in 96- and
384-well plates. See Note 14 for modified methods for infection of J774 and THP-1 cells.
10. On the day of infection, remove the medium and add 0.5 mL
of bacterial-uptake buffer suspension, prepared as described
above, to each well in a 24-well plate (see Note 15).
11. Incubate plates for 1 h at 37
C and 5% CO 2 .
12. Remove bacterial suspension and wash each well twice with
1 mL of pre-warmed wash medium.
13. Add 0.5 mL of fresh, pre-warmed macrophage infection
medium.
14. Incubate at 37
C and 5% CO 2 .
15. To maintain M. tuberculosis-infected macrophages, change the
medium with fresh, pre-warmed macrophage infection
medium every 2 days.
16. Observe macrophages under an inverted light microscope to
ascertain cell health. Survival assays can be continued through
14 days postinfection, although survival trends may become
variable at time points later than 9 days postinfection.
3.9 Infection of
Primary Bone MarrowDerived Macrophages
with M. tuberculosis in
96- and 384-Well
Plates
1. To infect macrophages with M. tuberculosis in a 96- or 384-well
plate, follow the recommended volumes and concentrations of
macrophages for a specific plate type as described in Note 11.
See Note 14 for modified methods for infection of J774 and
THP-1 cells.
2. Prepare M. tuberculosis as described in Subheading 3.8; however, after declumping and resuspending bacteria in uptake
buffer, adjust to 0.2 OD 600 with macrophage infection
medium.
3. For a 96-well plate, add 20 μL of the bacterial suspension to
each well, and for a 384-well plate, add 5 μL of the bacterial
suspension per well (do not remove medium prior to infection). The medium in the wells does not need to be changed or
washed during the infection, which can reliably proceed for
approximately 6 days (see Note 16).
Macrophage Infection Models for Mycobacterium tuberculosis
177