1.2 Intracellular
Activity
Human THP-1 Monocytes have been shown as a good model to
study the intracellular stages of M. tuberculosis [8, 9]. In this chapter, we also describe an assay to determine the effect of a compound
on the growth of H37Rv inside these monocytes. The assay uses a
luminescent strain of M. tuberculosis to infect the monocytes, and
luciferase activity is then measured using a commercial reagent
(Bright-Glo™) [10]. The reagent causes cell lysis and generates a
luminescent signal that is proportional to the activity of luciferase
present, which in turn is proportional to the number of viable cells
in the culture. The assay relies on the activity of a thermostable
luciferase and in the properties of a buffer formulation for extracting luciferase from bacteria.
2 Materials
2.1 Plate Preparation
1. 96- or 384-well V-bottom plates.
2. Compound to be tested at 10 mM in DMSO (Stock solution).
3. DMSO (see Note 2).
4. 0.1 μg/mL rifampicin.
5. Antitubercular standards. Use a range of standards such as
isoniazid or fluoroquinolones (see Note 3).
6. Syringe-driven Filter units with a membrane pore size of
0.22 μM (Ex. Millex-GV).
7. Automated drug dispenser (D300e Digital Dispenser, HP) (see
Note 4).
8. Electronic Multichannel Pipettes.
2.2 Preparation
of Inoculum
1. Mycobacterium strains of interest, e.g., M. tuberculosis H37Rv,
Mycobacterium bovis BCG Pasteur, Mycobacterium smegmatis
mc
2 155. For intracellular assay, we use the reporter strain
M. tuberculosis H37Rv:luc described in Sorrentino et al. [3].
2. 7H9-ADC-Tyloxapol: dissolve 4.7 g Middlebrook 7H9 broth
base in 900 mL deionized water, Add 5 mL of 10% w/v
Tyloxapol and 10% Albumin-Dextrose-Catalase (ADC) enrichment (Becton Dickinson).
3. 25 cm
2 cell culture flasks.
4. Spectrofluorimeter.
5. Multidrop™ Combi Reagent Dispenser and Multichannel
pipettes.
2.3 Measuring
Growth Using
Resazurin
1. 96- or 384-well, flat bottom Black plates.
2. Resazurin Solution: Dissolve one tablet of Resazurin (Resazurin Tablets for Milk Testing; VWR International Ltd) in
30 mL of sterile PBS. Sterilize by filtration (0.22 μm).
3. Fluorimeter, e.g., Spectramax M5.
640
Esther Pe ´ rez-Herra ´ n and Alfonso Mendoza
Activity
Human THP-1 Monocytes have been shown as a good model to
study the intracellular stages of M. tuberculosis [8, 9]. In this chapter, we also describe an assay to determine the effect of a compound
on the growth of H37Rv inside these monocytes. The assay uses a
luminescent strain of M. tuberculosis to infect the monocytes, and
luciferase activity is then measured using a commercial reagent
(Bright-Glo™) [10]. The reagent causes cell lysis and generates a
luminescent signal that is proportional to the activity of luciferase
present, which in turn is proportional to the number of viable cells
in the culture. The assay relies on the activity of a thermostable
luciferase and in the properties of a buffer formulation for extracting luciferase from bacteria.
2 Materials
2.1 Plate Preparation
1. 96- or 384-well V-bottom plates.
2. Compound to be tested at 10 mM in DMSO (Stock solution).
3. DMSO (see Note 2).
4. 0.1 μg/mL rifampicin.
5. Antitubercular standards. Use a range of standards such as
isoniazid or fluoroquinolones (see Note 3).
6. Syringe-driven Filter units with a membrane pore size of
0.22 μM (Ex. Millex-GV).
7. Automated drug dispenser (D300e Digital Dispenser, HP) (see
Note 4).
8. Electronic Multichannel Pipettes.
2.2 Preparation
of Inoculum
1. Mycobacterium strains of interest, e.g., M. tuberculosis H37Rv,
Mycobacterium bovis BCG Pasteur, Mycobacterium smegmatis
mc
2 155. For intracellular assay, we use the reporter strain
M. tuberculosis H37Rv:luc described in Sorrentino et al. [3].
2. 7H9-ADC-Tyloxapol: dissolve 4.7 g Middlebrook 7H9 broth
base in 900 mL deionized water, Add 5 mL of 10% w/v
Tyloxapol and 10% Albumin-Dextrose-Catalase (ADC) enrichment (Becton Dickinson).
3. 25 cm
2 cell culture flasks.
4. Spectrofluorimeter.
5. Multidrop™ Combi Reagent Dispenser and Multichannel
pipettes.
2.3 Measuring
Growth Using
Resazurin
1. 96- or 384-well, flat bottom Black plates.
2. Resazurin Solution: Dissolve one tablet of Resazurin (Resazurin Tablets for Milk Testing; VWR International Ltd) in
30 mL of sterile PBS. Sterilize by filtration (0.22 μm).
3. Fluorimeter, e.g., Spectramax M5.
640
Esther Pe ´ rez-Herra ´ n and Alfonso Mendoza