12. When robotically dispensing compounds using tips, make sure
to mix at least one time. Assay ready plates prepared by acoustically dispensing DMSO stocks of compound onto well bottoms of assay plates have worked well for HTS for our group.
However, the facility housing the compounds must be maintained under extraordinarily clean conditions to maintain the
sterility of the plates. When working with wild-type
M. tuberculosis, assay plates are processed in a sterile glove
box in the BSL3 facility. We always use filter tips to help
control mold.
13. Sometimes, a compound may precipitate or change color when
it is added to the nonreplicating M. tuberculosis cells, even
though the same compound was assumed to be soluble
because it had never precipitated in 7H9-ADN. Any time one
observes a color change or precipitation in nonreplicatingcomplete medium, the compound is probably chemically modified under these conditions (usually due to the acidic pH
and/or reactive nitrogen species from NaNO 2 ).
14. Controls can be added to columns 1, 2, 23, and 24. We prefer
columns 1 and 2 to contain DMSO as a vehicle control, while
column 23 contains oxyphenbutazone and Column 24 contains rifampicin. One can have less than 16 replicates for each
control compound, but a minimum of three wells are required
to obtain reasonable Z
0 scores. To date, one of the best
nonreplicating-specific compound controls is oxyphenbutazone, which requires acidic pH and NaNO 2 to kill
M. tuberculosis in the four-stress nonreplicating model
[49]. If oxyphenbutazone fails to kill nonreplicating
M. tuberculosis, ensure that the pH of the nonreplicating
medium is 5.0 and that 0.5 mM NaNO 2 was added. Because
a NaNO 2 stock may slowly degrade in water (ddH 2 O from
some sources may be acidic), it is important to use NaNO 2
prepared the same day and kept on ice until ready to use.
Alternatively, the preweighed NaNO 2 powder can be taken to
the BSL3 and the ddH 2 O added immediately before use.
Rifampicin can be used as a control for both replicating and
nonreplicating M. tuberculosis. Because the nonreplicating
activity of rifampicin is significantly less potent than its activity
against replicating M. tuberculosis, make sure to use rifampicin
at concentrations higher than the replicating MIC 90 . Isoniazid
is inactive in the nonreplicating model and serves as a negative
control for CFU-based assays. Due to its potent replicating
activity, INH will have some activity in the nonreplicating assay
by virtue of killing during the replicating outgrowth phase.
15. In addition to maintaining 1% O 2 , keeping the hypoxic chamber sealed will help prevent evaporation by maintaining high
humidity.
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Ben Gold et al.
to mix at least one time. Assay ready plates prepared by acoustically dispensing DMSO stocks of compound onto well bottoms of assay plates have worked well for HTS for our group.
However, the facility housing the compounds must be maintained under extraordinarily clean conditions to maintain the
sterility of the plates. When working with wild-type
M. tuberculosis, assay plates are processed in a sterile glove
box in the BSL3 facility. We always use filter tips to help
control mold.
13. Sometimes, a compound may precipitate or change color when
it is added to the nonreplicating M. tuberculosis cells, even
though the same compound was assumed to be soluble
because it had never precipitated in 7H9-ADN. Any time one
observes a color change or precipitation in nonreplicatingcomplete medium, the compound is probably chemically modified under these conditions (usually due to the acidic pH
and/or reactive nitrogen species from NaNO 2 ).
14. Controls can be added to columns 1, 2, 23, and 24. We prefer
columns 1 and 2 to contain DMSO as a vehicle control, while
column 23 contains oxyphenbutazone and Column 24 contains rifampicin. One can have less than 16 replicates for each
control compound, but a minimum of three wells are required
to obtain reasonable Z
0 scores. To date, one of the best
nonreplicating-specific compound controls is oxyphenbutazone, which requires acidic pH and NaNO 2 to kill
M. tuberculosis in the four-stress nonreplicating model
[49]. If oxyphenbutazone fails to kill nonreplicating
M. tuberculosis, ensure that the pH of the nonreplicating
medium is 5.0 and that 0.5 mM NaNO 2 was added. Because
a NaNO 2 stock may slowly degrade in water (ddH 2 O from
some sources may be acidic), it is important to use NaNO 2
prepared the same day and kept on ice until ready to use.
Alternatively, the preweighed NaNO 2 powder can be taken to
the BSL3 and the ddH 2 O added immediately before use.
Rifampicin can be used as a control for both replicating and
nonreplicating M. tuberculosis. Because the nonreplicating
activity of rifampicin is significantly less potent than its activity
against replicating M. tuberculosis, make sure to use rifampicin
at concentrations higher than the replicating MIC 90 . Isoniazid
is inactive in the nonreplicating model and serves as a negative
control for CFU-based assays. Due to its potent replicating
activity, INH will have some activity in the nonreplicating assay
by virtue of killing during the replicating outgrowth phase.
15. In addition to maintaining 1% O 2 , keeping the hypoxic chamber sealed will help prevent evaporation by maintaining high
humidity.
628
Ben Gold et al.