derived macrophages. We also describe protocols for preparing and
dispensing all the components involved in the various screening
methodologies, which include the distribution of infected cells
directly onto library compounds, and distribution and transfection
of siRNA for cell infection. We describe how macrophages are
prepared and infected at a low multiplicity of infection (MOI)
with an eGFP-expressing M. tuberculosis strain prior to incubation
with compounds. Particular attention is given to the description of
bacterial culture methods allowing the growth of non-clumping
bacteria and the use of two-week pre-cultures of mycobacteria, as
these parameters are crucial for successful and reproducible infections of macrophages.
Then, we describe cell staining procedures to monitor intracellular trafficking and replication of M. tuberculosis inside host cells,
either for end point studies when kinetic data are not needed, or for
dynamic studies requiring image acquisition for at least 3 days. We
propose a population-based, multi-parametric analysis pipeline that
can be used to monitor the intracellular fate of M. tuberculosis and
the dynamics of cellular events involved in host defense, such as
phagosomal maturation (acidification) and vacuolar rupture, generation of reactive oxygen species (RO) and nitric oxide
(NO) synthesis, a zinc poisoning system, and the investigation of
the accumulation of lipid droplets, also known as lipid bodies.
Finally, we outline basic image parameters that can best depict
the impact of the intervention (compounds or siRNA) on mycobacterial survival or virulence.
2 Materials
2.1 Growth
of Mycobacterium
tuberculosis Strains
for Cellular
Infection Assay
N.B. Requirement of a biosafety cabinet and a dedicated biosafety
laboratory adapted for pathogens of class 3.
1. Bacterial strains (see Note 1).
2. 20% w/v Tween 80: Add 20 g of Tween 80 in 100 mL of
deionized water. Filter-sterilize through a 0.22 μm cellulose
ester membrane.
3. Hygromycin B: 50 mg/mL stock solution (supplied ready to
use). Store at 4
C for 24 months from date of manufacture.
Add to medium at a final concentration of 50 μg/mL.
4. Kanamycin sulfate: store powder at 4
C. Prepare 25 mg/mL
stock in Dulbecco’s phosphate-buffered saline (DPBS). Store
at À20
C in 500 μL aliquots for up to 1 year. Add to medium
at a final concentration of 25 μg/mL.
5. Middlebrook OADC enrichment (supplied ready to use), containing oleic acid, bovine albumin, dextrose, catalase, and
NaCl. Store at 4
C in the dark.
652
Nathalie Deboosere et al.
dispensing all the components involved in the various screening
methodologies, which include the distribution of infected cells
directly onto library compounds, and distribution and transfection
of siRNA for cell infection. We describe how macrophages are
prepared and infected at a low multiplicity of infection (MOI)
with an eGFP-expressing M. tuberculosis strain prior to incubation
with compounds. Particular attention is given to the description of
bacterial culture methods allowing the growth of non-clumping
bacteria and the use of two-week pre-cultures of mycobacteria, as
these parameters are crucial for successful and reproducible infections of macrophages.
Then, we describe cell staining procedures to monitor intracellular trafficking and replication of M. tuberculosis inside host cells,
either for end point studies when kinetic data are not needed, or for
dynamic studies requiring image acquisition for at least 3 days. We
propose a population-based, multi-parametric analysis pipeline that
can be used to monitor the intracellular fate of M. tuberculosis and
the dynamics of cellular events involved in host defense, such as
phagosomal maturation (acidification) and vacuolar rupture, generation of reactive oxygen species (RO) and nitric oxide
(NO) synthesis, a zinc poisoning system, and the investigation of
the accumulation of lipid droplets, also known as lipid bodies.
Finally, we outline basic image parameters that can best depict
the impact of the intervention (compounds or siRNA) on mycobacterial survival or virulence.
2 Materials
2.1 Growth
of Mycobacterium
tuberculosis Strains
for Cellular
Infection Assay
N.B. Requirement of a biosafety cabinet and a dedicated biosafety
laboratory adapted for pathogens of class 3.
1. Bacterial strains (see Note 1).
2. 20% w/v Tween 80: Add 20 g of Tween 80 in 100 mL of
deionized water. Filter-sterilize through a 0.22 μm cellulose
ester membrane.
3. Hygromycin B: 50 mg/mL stock solution (supplied ready to
use). Store at 4
C for 24 months from date of manufacture.
Add to medium at a final concentration of 50 μg/mL.
4. Kanamycin sulfate: store powder at 4
C. Prepare 25 mg/mL
stock in Dulbecco’s phosphate-buffered saline (DPBS). Store
at À20
C in 500 μL aliquots for up to 1 year. Add to medium
at a final concentration of 25 μg/mL.
5. Middlebrook OADC enrichment (supplied ready to use), containing oleic acid, bovine albumin, dextrose, catalase, and
NaCl. Store at 4
C in the dark.
652
Nathalie Deboosere et al.