16. For the infection assays, it is recommended to maintain the
bacterial culture for up to 14 days before being used, in order
to reach the exponential phase of bacterial growth.
17. As the presence of aggregates in the mycobacterial inoculum
could lead to a heterogeneous invasion process, it is important
to carefully prepare the bacteria suspensions. To remove bacterial aggregates, centrifuge at 220 Â g for 2 min. You should see
a well dispersed suspension without aggregates. You may
repeat the centrifugation step if needed.
18. You can adapt the bacterial concentration to reach higher or
lower multiplicity of infection (MOI), as required. The MOI of
2 is generally used with macrophage cell lines. A MOI of 1 is
generally preferred with human and murine primary cells, but
MOI of 5 up to 20 are recommended for the infection of A549
epithelial cells.
19. While monocytes can be stored in liquid nitrogen for several
months, it is not recommended to freeze the isolated CD14
+
fraction as it could strongly decrease the yield of the overall
purification due to cell mortality.
20. An alternative way for infection is possible on adherent differentiated THP-1 macrophage cells. For this infection assay,
20 Â 10
6 cells in suspension in 20 mL of pre-warmed RPMIFBS is first differentiated with 50 ng/mL PMA in a 75 cm
2
flask and incubated for 3 days, as described in steps 8–10 in
Subheading 3.3. Remove the medium and add directly a bacteria suspension in pre-warmed RPMI-FBS on the cell monolayer to reach MOI ranged from 2 up to 5. Incubate for 2 h at
37
C, 5% CO 2 to allow macrophage infection. Wash and
detach the infected cells as described in steps 11–17 in Subheading 3.3. Centrifuge at 220 Â g for 10 min and suspend the
cell pellet in RPMI-FBS to adjust cell suspension to 5 Â 10
5
cells/mL. Dispense 40 μL of infected cells per well
(corresponding to 2 Â 10
4 THP-1 macrophages/well) and
follow the steps 16–21 in Subheading 3.12.
21. An alternative way for infection is possible: bacteria (20 μL)
and BMDM (30 μL) could be seeded together on the day of
experiment. 2 h are enough to allow macrophage adhesion.
22. An alternative assay is possible to identify new compounds
possibly involved in impairing or inducing of zinc accumulation inside of phagosome in macrophage. For this assay, add
directly 20 μL compounds on 40 μL cells on day 1. Incubate
assay plate at 37
C in 5% CO 2 for 4 h. Remove the supernatant
from the plates using a multichannel pipette. Infect cells by
adding 20 μL per well of H37Rv-RFP to have a final MOI of
1 in RPMI-FBS (see Subheading 3.1). And follow the steps 5–
19 in Subheading 3.22.
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Nathalie Deboosere et al.
bacterial culture for up to 14 days before being used, in order
to reach the exponential phase of bacterial growth.
17. As the presence of aggregates in the mycobacterial inoculum
could lead to a heterogeneous invasion process, it is important
to carefully prepare the bacteria suspensions. To remove bacterial aggregates, centrifuge at 220 Â g for 2 min. You should see
a well dispersed suspension without aggregates. You may
repeat the centrifugation step if needed.
18. You can adapt the bacterial concentration to reach higher or
lower multiplicity of infection (MOI), as required. The MOI of
2 is generally used with macrophage cell lines. A MOI of 1 is
generally preferred with human and murine primary cells, but
MOI of 5 up to 20 are recommended for the infection of A549
epithelial cells.
19. While monocytes can be stored in liquid nitrogen for several
months, it is not recommended to freeze the isolated CD14
+
fraction as it could strongly decrease the yield of the overall
purification due to cell mortality.
20. An alternative way for infection is possible on adherent differentiated THP-1 macrophage cells. For this infection assay,
20 Â 10
6 cells in suspension in 20 mL of pre-warmed RPMIFBS is first differentiated with 50 ng/mL PMA in a 75 cm
2
flask and incubated for 3 days, as described in steps 8–10 in
Subheading 3.3. Remove the medium and add directly a bacteria suspension in pre-warmed RPMI-FBS on the cell monolayer to reach MOI ranged from 2 up to 5. Incubate for 2 h at
37
C, 5% CO 2 to allow macrophage infection. Wash and
detach the infected cells as described in steps 11–17 in Subheading 3.3. Centrifuge at 220 Â g for 10 min and suspend the
cell pellet in RPMI-FBS to adjust cell suspension to 5 Â 10
5
cells/mL. Dispense 40 μL of infected cells per well
(corresponding to 2 Â 10
4 THP-1 macrophages/well) and
follow the steps 16–21 in Subheading 3.12.
21. An alternative way for infection is possible: bacteria (20 μL)
and BMDM (30 μL) could be seeded together on the day of
experiment. 2 h are enough to allow macrophage adhesion.
22. An alternative assay is possible to identify new compounds
possibly involved in impairing or inducing of zinc accumulation inside of phagosome in macrophage. For this assay, add
directly 20 μL compounds on 40 μL cells on day 1. Incubate
assay plate at 37
C in 5% CO 2 for 4 h. Remove the supernatant
from the plates using a multichannel pipette. Infect cells by
adding 20 μL per well of H37Rv-RFP to have a final MOI of
1 in RPMI-FBS (see Subheading 3.1). And follow the steps 5–
19 in Subheading 3.22.
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Nathalie Deboosere et al.