3. Wash cells with 50 μL per well of RPMI-FBS using a multichannel pipette.
4. Repeat steps 2 and 3.
5. Add 50 μL per well of (1) a staining solution in RPMI-FBS
containing 10 μg/mL Hoechst 33342 and 5 μg/mL CellMask
Deep Red Plasmas Membrane Stain or (2) a staining/fixative
solution, obtained by diluting 10 μg/mL Hoechst 33342 or
DAPI solution in 4% formaldehyde solution.
6. Incubate for 30 min at 37
C, 5% CO 2 .
7. For (1) stained live cells, immediately proceed to image acquisition without washing or add 50 μL per well of 4% formaldehyde solution, incubate for 30 min at room temperature and
wash cells twice with DPBS, before proceeding with image
acquisition using the high-content confocal microscope (see
Subheading 3.25). Use 20Â or 60Â magnification. After fixation, plates can be stored for up to one month, sealed and kept
in the dark at 4
C.
8. For (2) stained and fixed cells, wash cells twice with 50 μL of
DPBS, before proceeding with image acquisition, using the
high-content confocal microscope (see Subheading 3.25). Use
20Â or 60Â magnification. Plates can be stored.
3.17 Staining of Cell
Nucleus, Cytoplasm
and/or F-Actin
on Fixed and
Permeabilized Cells
If you do not want to proceed immediately with staining for immunofluorescence assay, a fixation step is required prior to staining (see
Fig. 5a).
1. Perform the intracellular mycobacterial replication assays (see
Subheadings 3.11–3.14).
2. Carefully remove the supernatant from the plates using a multichannel pipette and avoid to disturb the cell layer.
3. Wash cells with 50 μL per well of RPMI-FBS using a multichannel pipette.
4. Repeat steps 2 and 3.
5. Add 50 μL per well of 4% formaldehyde solution used as
fixative solution.
6. Incubate for 30 min at room temperature.
7. Repeat twice steps 2 and 3 with DPBS.
8. Add 50 μL per well of 0.1% w/v Triton X-100 solution in
DPBS, used as permeabilization solution.
9. Incubate for 5 min at room temperature.
10. Repeat twice steps 2 and 3 with DPBS.
11. Add 25 μL per well of a staining solution in DPBS containing
(1) 10 μg/mL Hoechst 33342 and 2 μg/mL CellMask Blue
Stain or (2) 1.65 μM Alexa Fluor 660 Phalloidin staining
solution.
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Nathalie Deboosere et al.
4. Repeat steps 2 and 3.
5. Add 50 μL per well of (1) a staining solution in RPMI-FBS
containing 10 μg/mL Hoechst 33342 and 5 μg/mL CellMask
Deep Red Plasmas Membrane Stain or (2) a staining/fixative
solution, obtained by diluting 10 μg/mL Hoechst 33342 or
DAPI solution in 4% formaldehyde solution.
6. Incubate for 30 min at 37
C, 5% CO 2 .
7. For (1) stained live cells, immediately proceed to image acquisition without washing or add 50 μL per well of 4% formaldehyde solution, incubate for 30 min at room temperature and
wash cells twice with DPBS, before proceeding with image
acquisition using the high-content confocal microscope (see
Subheading 3.25). Use 20Â or 60Â magnification. After fixation, plates can be stored for up to one month, sealed and kept
in the dark at 4
C.
8. For (2) stained and fixed cells, wash cells twice with 50 μL of
DPBS, before proceeding with image acquisition, using the
high-content confocal microscope (see Subheading 3.25). Use
20Â or 60Â magnification. Plates can be stored.
3.17 Staining of Cell
Nucleus, Cytoplasm
and/or F-Actin
on Fixed and
Permeabilized Cells
If you do not want to proceed immediately with staining for immunofluorescence assay, a fixation step is required prior to staining (see
Fig. 5a).
1. Perform the intracellular mycobacterial replication assays (see
Subheadings 3.11–3.14).
2. Carefully remove the supernatant from the plates using a multichannel pipette and avoid to disturb the cell layer.
3. Wash cells with 50 μL per well of RPMI-FBS using a multichannel pipette.
4. Repeat steps 2 and 3.
5. Add 50 μL per well of 4% formaldehyde solution used as
fixative solution.
6. Incubate for 30 min at room temperature.
7. Repeat twice steps 2 and 3 with DPBS.
8. Add 50 μL per well of 0.1% w/v Triton X-100 solution in
DPBS, used as permeabilization solution.
9. Incubate for 5 min at room temperature.
10. Repeat twice steps 2 and 3 with DPBS.
11. Add 25 μL per well of a staining solution in DPBS containing
(1) 10 μg/mL Hoechst 33342 and 2 μg/mL CellMask Blue
Stain or (2) 1.65 μM Alexa Fluor 660 Phalloidin staining
solution.
682
Nathalie Deboosere et al.