3.20 Phagosomal
Vacuole Rupture Assay
M. tuberculosis translocates from the phagosome to the cytosol in
macrophages, involving the rupture of the enclosing phagosomal
membrane and coinciding with cell death by necrosis. The CCF4AM dye (Invitrogen) is cell permeable, β-lactamase substrate displaying a fluorescence resonance energy transfer (FRET) property.
In the presence of β-lactamase enzymatic activity in the cell cytoplasm, the CCF4-AM substrate is rapidly converted into its negatively charged metabolite CCF4, which corresponds to a shift in the
emission wavelength from green to blue. As M. tuberculosis
expresses membrane-associated β-lactamase BlaC, this dye can be
used to characterize the ability of mycobacteria to escape their
phagosomal vacuoles and reach the cytoplasm. The green intensity
(CCF4-AM) decreases while the blue intensity (CCF4) increases in
cells infected with live-mycobacteria, whereas cells infected with
heat-killed mycobacteria do not show such changes.
1. Seed 4 Â 10
4 cells THP-1 macrophages (see Subheading 3.3) in
30 μL per well of RPMI-FBS in 384-well plates.
2. Incubate plates overnight at 37
C with 5% CO 2 .
3. One hour before infection, in dedicated infected and
non-infected wells for positive controls (vacuole rupture):
remove medium and add 50 μL per well of 20 nM Bafilomycin
in RPMI-FBS (see Note 12).
4. Remove the supernatant from all wells of the plates using a
multichannel pipette.
5. In dedicated non-infected wells for negative controls
(no vacuolar rupture): add 50 μL per well of heat-killed
H37Rv-RFP bacteria at a MOI of 2.
6. In dedicated wells for cell infection: add 50 μL per well of
H37Rv-RFP bacteria at a MOI of 2 in RPMI-FBS (see Subheading 3.1 and Note 18).
7. Seal plates using breathable sealing films and centrifuge
220 Â g for 1 min at room temperature.
8. Incubate plates for 2 h at 37
C with 5% CO 2 .
9. Remove the breathable sealing films.
10. Remove the supernatant from the plates using a multichannel
pipette.
11. Wash cells with 50 μL per well of RPMI-FBS using a multichannel pipette.
12. Add 50 μL per well of RPMI-FBS containing 50 μg/mL
amikacin.
13. Incubate plates for 1 h at 37
C with 5% CO 2 .
14. Repeat twice steps 10 and 11.
15. Incubate plates overnight at 37
C with 5% CO 2 .
686
Nathalie Deboosere et al.
Vacuole Rupture Assay
M. tuberculosis translocates from the phagosome to the cytosol in
macrophages, involving the rupture of the enclosing phagosomal
membrane and coinciding with cell death by necrosis. The CCF4AM dye (Invitrogen) is cell permeable, β-lactamase substrate displaying a fluorescence resonance energy transfer (FRET) property.
In the presence of β-lactamase enzymatic activity in the cell cytoplasm, the CCF4-AM substrate is rapidly converted into its negatively charged metabolite CCF4, which corresponds to a shift in the
emission wavelength from green to blue. As M. tuberculosis
expresses membrane-associated β-lactamase BlaC, this dye can be
used to characterize the ability of mycobacteria to escape their
phagosomal vacuoles and reach the cytoplasm. The green intensity
(CCF4-AM) decreases while the blue intensity (CCF4) increases in
cells infected with live-mycobacteria, whereas cells infected with
heat-killed mycobacteria do not show such changes.
1. Seed 4 Â 10
4 cells THP-1 macrophages (see Subheading 3.3) in
30 μL per well of RPMI-FBS in 384-well plates.
2. Incubate plates overnight at 37
C with 5% CO 2 .
3. One hour before infection, in dedicated infected and
non-infected wells for positive controls (vacuole rupture):
remove medium and add 50 μL per well of 20 nM Bafilomycin
in RPMI-FBS (see Note 12).
4. Remove the supernatant from all wells of the plates using a
multichannel pipette.
5. In dedicated non-infected wells for negative controls
(no vacuolar rupture): add 50 μL per well of heat-killed
H37Rv-RFP bacteria at a MOI of 2.
6. In dedicated wells for cell infection: add 50 μL per well of
H37Rv-RFP bacteria at a MOI of 2 in RPMI-FBS (see Subheading 3.1 and Note 18).
7. Seal plates using breathable sealing films and centrifuge
220 Â g for 1 min at room temperature.
8. Incubate plates for 2 h at 37
C with 5% CO 2 .
9. Remove the breathable sealing films.
10. Remove the supernatant from the plates using a multichannel
pipette.
11. Wash cells with 50 μL per well of RPMI-FBS using a multichannel pipette.
12. Add 50 μL per well of RPMI-FBS containing 50 μg/mL
amikacin.
13. Incubate plates for 1 h at 37
C with 5% CO 2 .
14. Repeat twice steps 10 and 11.
15. Incubate plates overnight at 37
C with 5% CO 2 .
686
Nathalie Deboosere et al.