8. The volume of compound to be added to the assay plate
depends on the concentration of the master plate and the
concentration required for the assay. This protocol assumes a
1:100 dilution from master plate to assay plate and a final
culture volume of 25 μL per well in a 384-well plate. The
maximum concentration of DMSO that allows viability of
mycobacteria is 5% but only 0.5% for monocyte cell cultures.
For luminescence, DMSO may affect the signal depending on
the luminometer. Therefore, adjusting the concentration in the
master plate to accommodate the addition of a larger volume of
the DMSO into the assay plate will not work. In such cases,
prepare an intermediary plate in which the compound(s) are
diluted into culture medium. Critically the concentration of
the solvent must be maintained at or below the level that it is
toxic for the mycobacteria or the THP1 cell.
9. Stocks of bacteria can be kept in 15–20% glycerol at À80
C.
Passaging of bacterial cultures should be minimized to avoid
selection and accumulation of spontaneous mutations.
10. A Ziehl-Neelsen stain can be useful to check the purity of the
culture or plate the culture in non-selective agar plates such as
chocolate blood agar or LB agar.
11. Mycobacteria can be cultured in flasks without shaking, in
roller bottles or in a glass vessel with shaking. Due to biosafety,
a cell culture flask is the best option or for larger volumes,
plastic roller bottles.
12. It is important to determine the correlation between OD and
CFU/mL. The most important factors in this ratio are the
culture medium, culture conditions, and the absorbance
reader. In our case, an OD 600 of 0.125 is equivalent to
1 Â 10
7 CFU/mL.
13. The difference between positive and negative controls (signal/
noise) for resazurin read- 384-well plates is 4–8 so we generally
avoid its use.
14. Plates can also be read visually. The MIC value is the compound concentration in the first blue well, i.e., the minimum
concentration of compound that completely inhibits visible
growth.
15. Compounds can directly interfere with the read–out by reducing the resazurin. In this case, the MIC is calculated as the
concentration at which the fluorescent signal reaches a plateau.
16. ATP is measured using a commercial reagent (BacTiter-Glo™
Microbial Cell Viability Assay from Promega). The reagent
causes bacterial cell lysis and generates a luminescent signal
which is proportional to the amount of ATP present. The
assay relies on the activity of a thermostable luciferase and the
646
Esther Pe ´ rez-Herra ´ n and Alfonso Mendoza
depends on the concentration of the master plate and the
concentration required for the assay. This protocol assumes a
1:100 dilution from master plate to assay plate and a final
culture volume of 25 μL per well in a 384-well plate. The
maximum concentration of DMSO that allows viability of
mycobacteria is 5% but only 0.5% for monocyte cell cultures.
For luminescence, DMSO may affect the signal depending on
the luminometer. Therefore, adjusting the concentration in the
master plate to accommodate the addition of a larger volume of
the DMSO into the assay plate will not work. In such cases,
prepare an intermediary plate in which the compound(s) are
diluted into culture medium. Critically the concentration of
the solvent must be maintained at or below the level that it is
toxic for the mycobacteria or the THP1 cell.
9. Stocks of bacteria can be kept in 15–20% glycerol at À80
C.
Passaging of bacterial cultures should be minimized to avoid
selection and accumulation of spontaneous mutations.
10. A Ziehl-Neelsen stain can be useful to check the purity of the
culture or plate the culture in non-selective agar plates such as
chocolate blood agar or LB agar.
11. Mycobacteria can be cultured in flasks without shaking, in
roller bottles or in a glass vessel with shaking. Due to biosafety,
a cell culture flask is the best option or for larger volumes,
plastic roller bottles.
12. It is important to determine the correlation between OD and
CFU/mL. The most important factors in this ratio are the
culture medium, culture conditions, and the absorbance
reader. In our case, an OD 600 of 0.125 is equivalent to
1 Â 10
7 CFU/mL.
13. The difference between positive and negative controls (signal/
noise) for resazurin read- 384-well plates is 4–8 so we generally
avoid its use.
14. Plates can also be read visually. The MIC value is the compound concentration in the first blue well, i.e., the minimum
concentration of compound that completely inhibits visible
growth.
15. Compounds can directly interfere with the read–out by reducing the resazurin. In this case, the MIC is calculated as the
concentration at which the fluorescent signal reaches a plateau.
16. ATP is measured using a commercial reagent (BacTiter-Glo™
Microbial Cell Viability Assay from Promega). The reagent
causes bacterial cell lysis and generates a luminescent signal
which is proportional to the amount of ATP present. The
assay relies on the activity of a thermostable luciferase and the
646
Esther Pe ´ rez-Herra ´ n and Alfonso Mendoza