5. Seal plates using breathable sealing films and centrifuge
220 Â g for 1 min at room temperature.
6. Incubate plates for 4 h at 37
C with 5% CO 2 .
7. Remove the breathable sealing films.
8. Remove the supernatant from the plates using a multichannel
pipette.
9. Wash cells three times with 50 μL per well of DPBS using a
multichannel pipette.
10. Add 50 μL per well of 4% formaldehyde fixative solution.
11. Incubate for 30 min at room temperature.
12. Repeat twice steps 8 and 9 with DPBS.
13. Add 25 μL per well of the 2 μM FluoZin-3 staining solution
in DPBS.
14. In dedicated wells for positive controls (intracellular Zinc indicator controls): add 10 μL per well indicator salt suspensions
ranging from 10 to 100 μM (or directly into the 2 μM FluoZin3 staining solution to obtain the recommended final concentration of 10–100 μM).
15. Incubate plates for 50 min at room temperature in the dark
(cover the plate to protect from light and evaporation).
16. Add 25 μL per well of a nucleus staining solution,
corresponding to a 10 μg/mL DAPI solution to have a final
concentration of 5 μg/mL.
17. Incubate for 10 min at room temperature.
18. Repeat twice steps 8 and 9 with DPBS-FBS.
19. Immediately proceed to image the cells in 50 μL per well of
DPBS-FBS, using the high-content confocal microscope (see
Subheading 3.25). Use 60Â magnification. Plates cannot be
stored.
3.24 Lipid Body
Accumulation Assay
The occurrence of lipid body-loaded host cells during
M. tuberculosis infection, also called foamy macrophages, has been
observed in vivo within human granulomas and in vitro using
macrophages subjected to various stimuli. The accumulation of
cytoplasmic lipid bodies (LBs) was detected using the LipidTox
dye (Invitrogen), which labels neutral lipids. Following
M. tuberculosis infection, the number of lipid droplets per cell
increases. This assay is dedicated to the study of LB accumulation
and could lead to the identification of potential druggable targets.
1. Seed 2 Â 10
4 BMDM or 4 Â 10
4 BMDC (see Subheadings 3.8
and 3.9) in 40 μL per well of RPMI-FBS supplemented with
10% M-CSF or GM-CSF in 384-well plates.
2. Incubate overnight at 37
C with 5% CO 2 .
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Nathalie Deboosere et al.
220 Â g for 1 min at room temperature.
6. Incubate plates for 4 h at 37
C with 5% CO 2 .
7. Remove the breathable sealing films.
8. Remove the supernatant from the plates using a multichannel
pipette.
9. Wash cells three times with 50 μL per well of DPBS using a
multichannel pipette.
10. Add 50 μL per well of 4% formaldehyde fixative solution.
11. Incubate for 30 min at room temperature.
12. Repeat twice steps 8 and 9 with DPBS.
13. Add 25 μL per well of the 2 μM FluoZin-3 staining solution
in DPBS.
14. In dedicated wells for positive controls (intracellular Zinc indicator controls): add 10 μL per well indicator salt suspensions
ranging from 10 to 100 μM (or directly into the 2 μM FluoZin3 staining solution to obtain the recommended final concentration of 10–100 μM).
15. Incubate plates for 50 min at room temperature in the dark
(cover the plate to protect from light and evaporation).
16. Add 25 μL per well of a nucleus staining solution,
corresponding to a 10 μg/mL DAPI solution to have a final
concentration of 5 μg/mL.
17. Incubate for 10 min at room temperature.
18. Repeat twice steps 8 and 9 with DPBS-FBS.
19. Immediately proceed to image the cells in 50 μL per well of
DPBS-FBS, using the high-content confocal microscope (see
Subheading 3.25). Use 60Â magnification. Plates cannot be
stored.
3.24 Lipid Body
Accumulation Assay
The occurrence of lipid body-loaded host cells during
M. tuberculosis infection, also called foamy macrophages, has been
observed in vivo within human granulomas and in vitro using
macrophages subjected to various stimuli. The accumulation of
cytoplasmic lipid bodies (LBs) was detected using the LipidTox
dye (Invitrogen), which labels neutral lipids. Following
M. tuberculosis infection, the number of lipid droplets per cell
increases. This assay is dedicated to the study of LB accumulation
and could lead to the identification of potential druggable targets.
1. Seed 2 Â 10
4 BMDM or 4 Â 10
4 BMDC (see Subheadings 3.8
and 3.9) in 40 μL per well of RPMI-FBS supplemented with
10% M-CSF or GM-CSF in 384-well plates.
2. Incubate overnight at 37
C with 5% CO 2 .
690
Nathalie Deboosere et al.