7. Add 0.5 mM NaNO 2 from 100 mM stock to appropriate tubes
described in Table 2.
8. For each set of cells in a single deconvolution medium, pour
cells into a reagent reservoir and dispense 200 μL cells per well
in two 96-well plates using a 12-channel multichannel pipette.
9. Add test agents and DMSO controls as described in Subheading 3.3, steps 2 and 3 to all 16 microplates.
10. Place one set of duplicate microplates (eight plates, media
A-H) into the hypoxic chamber set at 1% O 2 and 5% CO 2
and place the other replicate set (eight plates, media A-H)
into an incubator set at 20% O 2 and 5% CO 2 (Table 2).
11. On day 3, set up standard outgrowths for all plates as described
in Subheading 3.3, steps 6–14.
12. Read OD 580 at day 7–10.
3.6 Identification
of NaNO 2 -Dependent
Compounds
If the compound’s activity on nonreplicating M. tuberculosis is
determined to be NaNO 2 -dependent (see Note 28), one can then
explore if the compound gains activity against replicating
M. tuberculosis in the presence of an NO donor at neutral pH,
such as DETA-NO. In contrast to the constant flux of NO and
reactive nitrogen species formed by NaNO 2 at an acidic pH, the
short half-life of DETA-NO at neutral pH (~22 h at pH 7.4 at RT)
requires daily addition.
1. Prepare a single-cell suspension of mid-log M. tuberculosis in
strain-appropriate 7H9 complete medium pH 6.6 at a density
of OD 580 ¼ 0.05 as described in Subheading 3.4, steps 1 and
2 (see Note 9). Determine inoculum as described in Subheading 3.4, step 3 by plating serial dilutions on 7H11-OADC agar
plates.
2. Dispense 200 μL of cells/well in 96-well plates.
3. Add 2 μL of 100Â stock solutions of test compounds and
mix well.
4. Add 2 μL of the 100Â DETA-NO stock solutions to appropriate wells approximately every 24 h (see Note 29). DETA-NO
concentrations to test are 0, 100, 200, and 400 μM.
5. Add 2 μL of 0.01 N NaOH as a negative control.
6. Add 2 μL of 100Â DETA as a negative control (see Note 30).
7. Plate for CFUs at the desired time points as described in
Subheading 3.4, steps 10–14. Include 10
À6 and 10
À7 dilutions for wells containing vehicle control and those in which
there was an obvious increase in optical density.
624
Ben Gold et al.
described in Table 2.
8. For each set of cells in a single deconvolution medium, pour
cells into a reagent reservoir and dispense 200 μL cells per well
in two 96-well plates using a 12-channel multichannel pipette.
9. Add test agents and DMSO controls as described in Subheading 3.3, steps 2 and 3 to all 16 microplates.
10. Place one set of duplicate microplates (eight plates, media
A-H) into the hypoxic chamber set at 1% O 2 and 5% CO 2
and place the other replicate set (eight plates, media A-H)
into an incubator set at 20% O 2 and 5% CO 2 (Table 2).
11. On day 3, set up standard outgrowths for all plates as described
in Subheading 3.3, steps 6–14.
12. Read OD 580 at day 7–10.
3.6 Identification
of NaNO 2 -Dependent
Compounds
If the compound’s activity on nonreplicating M. tuberculosis is
determined to be NaNO 2 -dependent (see Note 28), one can then
explore if the compound gains activity against replicating
M. tuberculosis in the presence of an NO donor at neutral pH,
such as DETA-NO. In contrast to the constant flux of NO and
reactive nitrogen species formed by NaNO 2 at an acidic pH, the
short half-life of DETA-NO at neutral pH (~22 h at pH 7.4 at RT)
requires daily addition.
1. Prepare a single-cell suspension of mid-log M. tuberculosis in
strain-appropriate 7H9 complete medium pH 6.6 at a density
of OD 580 ¼ 0.05 as described in Subheading 3.4, steps 1 and
2 (see Note 9). Determine inoculum as described in Subheading 3.4, step 3 by plating serial dilutions on 7H11-OADC agar
plates.
2. Dispense 200 μL of cells/well in 96-well plates.
3. Add 2 μL of 100Â stock solutions of test compounds and
mix well.
4. Add 2 μL of the 100Â DETA-NO stock solutions to appropriate wells approximately every 24 h (see Note 29). DETA-NO
concentrations to test are 0, 100, 200, and 400 μM.
5. Add 2 μL of 0.01 N NaOH as a negative control.
6. Add 2 μL of 100Â DETA as a negative control (see Note 30).
7. Plate for CFUs at the desired time points as described in
Subheading 3.4, steps 10–14. Include 10
À6 and 10
À7 dilutions for wells containing vehicle control and those in which
there was an obvious increase in optical density.
624
Ben Gold et al.