dilute the 5 mM SYTO 60 solution 1:200 in appropriate
complete medium to prepare 25 μM SYTO 60 solution.
(2) Directly add the 25 μM SYTO 60 1:5 into the volume of
medium contained in wells of 384-well assay plate to obtain a
final concentration of 5 μM SYTO™ 60 per well.
2.13 Intracellular
Mycobacterial
Replication Assays
for Mutant Screening
N.B. Requirement of a sterile environment, such as a biosafety
cabinet in a dedicated laboratory.
1. See items 10–16 under Subheading 2.11.
2. 50 mg/mL Amikacin stock solution in PBS (see Subheading
2.12, item 7).
2.14 Intracellular
Mycobacterial Uptake
and Replication
Assays
N.B. Requirement of a biosafety cabinet in a dedicated laboratory.
1. See items 11–16 under Subheading 2.11.
2. 50 mg/mL Amikacin stock solution in PBS (see Subheading
2.12, item 7).
2.15 Staining of Cell
Nucleus on Live Cells
for Dynamic Studies
1. See items 14 and 15 under Subheading 2.11.
2. 10 mg/mL Hoechst 33342 solution: store at 4
C and protect
from light. Freshly dilute the 10 μg/mL Hoechst 33342 solution 1:40 in appropriate complete medium to make a 250 ng/
mL Hoechst solution.
2.16 Staining of Cell
Nucleus and Plasma
Membrane on Live/
Fixed Cells
1. 10% Neutral buffered formalin solution (containing 4% formaldehyde): store at room temperature and use within 1 month
after opening.
2. 10 mg/mL Hoechst 33342 solution: store at 4
C and protect
from light. Freshly dilute the 10 mg/mL Hoechst 33342
solution 1:1000 in appropriate complete medium or formalin
solution to make a 10 μg/mL solution.
3. 4
0 ,6-diamidino-2-phenylindole (DAPI): store powder at 4
C
and protect from light. Prepare 1 mg/mL DAPI solution by
mixing 10 mg of DAPI in 10 mL of PBS. Filter-sterilize
through a 0.22 μm membrane. Store 500 μL aliquots at
À20
C. For assay, freshly dilute the 1 mg/mL DAPI solution
1:1000 in DPBS or 10% formalin solution to make a 1 μg/mL
working solution.
4. 5 mg/mL CellMask Deep Red Plasma Membrane Stain (Life
Technologies). Store at À20
C and protect from light. For
appropriate assay, freshly dilute 5 mg/mL CellMask Plasma
Membrane 1:1000 in RPMI-FBS to obtain 5 μg/mL CellMask
Deep Red Plasma Membrane Stain solution.
5. DPBS, no calcium, no magnesium, supplemented with 1% FBS
(DPBS-FBS): add 5 mL of FBS in a 500 mL DPBS bottle.
Store at 4
C for up to 1 month.
658
Nathalie Deboosere et al.
complete medium to prepare 25 μM SYTO 60 solution.
(2) Directly add the 25 μM SYTO 60 1:5 into the volume of
medium contained in wells of 384-well assay plate to obtain a
final concentration of 5 μM SYTO™ 60 per well.
2.13 Intracellular
Mycobacterial
Replication Assays
for Mutant Screening
N.B. Requirement of a sterile environment, such as a biosafety
cabinet in a dedicated laboratory.
1. See items 10–16 under Subheading 2.11.
2. 50 mg/mL Amikacin stock solution in PBS (see Subheading
2.12, item 7).
2.14 Intracellular
Mycobacterial Uptake
and Replication
Assays
N.B. Requirement of a biosafety cabinet in a dedicated laboratory.
1. See items 11–16 under Subheading 2.11.
2. 50 mg/mL Amikacin stock solution in PBS (see Subheading
2.12, item 7).
2.15 Staining of Cell
Nucleus on Live Cells
for Dynamic Studies
1. See items 14 and 15 under Subheading 2.11.
2. 10 mg/mL Hoechst 33342 solution: store at 4
C and protect
from light. Freshly dilute the 10 μg/mL Hoechst 33342 solution 1:40 in appropriate complete medium to make a 250 ng/
mL Hoechst solution.
2.16 Staining of Cell
Nucleus and Plasma
Membrane on Live/
Fixed Cells
1. 10% Neutral buffered formalin solution (containing 4% formaldehyde): store at room temperature and use within 1 month
after opening.
2. 10 mg/mL Hoechst 33342 solution: store at 4
C and protect
from light. Freshly dilute the 10 mg/mL Hoechst 33342
solution 1:1000 in appropriate complete medium or formalin
solution to make a 10 μg/mL solution.
3. 4
0 ,6-diamidino-2-phenylindole (DAPI): store powder at 4
C
and protect from light. Prepare 1 mg/mL DAPI solution by
mixing 10 mg of DAPI in 10 mL of PBS. Filter-sterilize
through a 0.22 μm membrane. Store 500 μL aliquots at
À20
C. For assay, freshly dilute the 1 mg/mL DAPI solution
1:1000 in DPBS or 10% formalin solution to make a 1 μg/mL
working solution.
4. 5 mg/mL CellMask Deep Red Plasma Membrane Stain (Life
Technologies). Store at À20
C and protect from light. For
appropriate assay, freshly dilute 5 mg/mL CellMask Plasma
Membrane 1:1000 in RPMI-FBS to obtain 5 μg/mL CellMask
Deep Red Plasma Membrane Stain solution.
5. DPBS, no calcium, no magnesium, supplemented with 1% FBS
(DPBS-FBS): add 5 mL of FBS in a 500 mL DPBS bottle.
Store at 4
C for up to 1 month.
658
Nathalie Deboosere et al.