the role of unknown host genes in the control of pathogens.
Conditionally immortalized myeloid progenitors from the bone
marrow driven by an estradiol-inducible HoxB8 further allows
the unlimited propagation of primary bone marrow-derived macrophages that can decrease experimental variability and decrease animal usage. Alternatively, immortalized cell-lines can be used to
study M. tuberculosis–macrophage interactions, including the
J774 and THP-1 cell lines [2, 3]. Additionally, alveolar
macrophage-like cells derived from the fetal liver, termed Max
Planck Institute (MPI) cells, can serve as a model of lung-specific
macrophage populations [4]. The response of M. tuberculosis to
intracellular environments can vary greatly depending on the macrophage cell type chosen [5], primary or immortalized, human or
murine; therefore, care should be taken when interpreting data
from an individual cell type. M. tuberculosis macrophage infections
can be used for a variety of purposes including characterization of:
(1) mutant virulence, (2) antimicrobial efficacy, (3) microbial gene
expression in response to immune pressure, (4) intracellular trafficking of microbes and host markers, and (5) the role of macrophage genes in the control of bacterial growth. Additionally,
macrophage infection models are amenable to 96-well and
384-well plate formats, enabling high throughput assays of
M. tuberculosis surivival when using fluorescent or luminescent
M. tuberculosis reporter strains. In this chapter, we describe methods (1) to isolate and propagate primary murine BMDMs, conditionally immortalized myeloid progenitor cells using HoxB8, Max
Planck Institute Alverolar macrophage-like cells, and the J774 and
THP-1 macrophage-like cell lines, (2) to infect macrophages with
M. tuberculosis, (3) to assay M. tuberculosis survival in macrophages,
and (4) to prepare infected macrophages for imaging.
2 Materials
2.1 Isolation of Bone
Marrow from Mice
1. Bone marrow macrophage medium (500 mL) (see Note 1):
335 mL Dulbecco’s Modified Eagle Medium (DMEM) with
4.5 g/L glucose and without sodium pyruvate and L-glutamine, 5 mL 100 mM sodium pyruvate, 5 mL 200 mM Lglutamine, 5 mL 100Â penicillin (10,000 I.U./mL) streptomycin (10,000 μg/mL), 50 mL heat inactivated,
low-endotoxin fetal bovine serum (FBS) (e.g., Hyclone™
FBS Thermo Scientific, Waltham, MA), 100 mL L-cell
conditioned medium (see Note 2). Sterilize by filtering
through a 0.22 μm filter.
2. DMEM with 4.5 g/L glucose and without sodium pyruvate
and L-glutamine.
3. 100 mm petri dishes.
168
Benjamin K. Johnson et al.
Conditionally immortalized myeloid progenitors from the bone
marrow driven by an estradiol-inducible HoxB8 further allows
the unlimited propagation of primary bone marrow-derived macrophages that can decrease experimental variability and decrease animal usage. Alternatively, immortalized cell-lines can be used to
study M. tuberculosis–macrophage interactions, including the
J774 and THP-1 cell lines [2, 3]. Additionally, alveolar
macrophage-like cells derived from the fetal liver, termed Max
Planck Institute (MPI) cells, can serve as a model of lung-specific
macrophage populations [4]. The response of M. tuberculosis to
intracellular environments can vary greatly depending on the macrophage cell type chosen [5], primary or immortalized, human or
murine; therefore, care should be taken when interpreting data
from an individual cell type. M. tuberculosis macrophage infections
can be used for a variety of purposes including characterization of:
(1) mutant virulence, (2) antimicrobial efficacy, (3) microbial gene
expression in response to immune pressure, (4) intracellular trafficking of microbes and host markers, and (5) the role of macrophage genes in the control of bacterial growth. Additionally,
macrophage infection models are amenable to 96-well and
384-well plate formats, enabling high throughput assays of
M. tuberculosis surivival when using fluorescent or luminescent
M. tuberculosis reporter strains. In this chapter, we describe methods (1) to isolate and propagate primary murine BMDMs, conditionally immortalized myeloid progenitor cells using HoxB8, Max
Planck Institute Alverolar macrophage-like cells, and the J774 and
THP-1 macrophage-like cell lines, (2) to infect macrophages with
M. tuberculosis, (3) to assay M. tuberculosis survival in macrophages,
and (4) to prepare infected macrophages for imaging.
2 Materials
2.1 Isolation of Bone
Marrow from Mice
1. Bone marrow macrophage medium (500 mL) (see Note 1):
335 mL Dulbecco’s Modified Eagle Medium (DMEM) with
4.5 g/L glucose and without sodium pyruvate and L-glutamine, 5 mL 100 mM sodium pyruvate, 5 mL 200 mM Lglutamine, 5 mL 100Â penicillin (10,000 I.U./mL) streptomycin (10,000 μg/mL), 50 mL heat inactivated,
low-endotoxin fetal bovine serum (FBS) (e.g., Hyclone™
FBS Thermo Scientific, Waltham, MA), 100 mL L-cell
conditioned medium (see Note 2). Sterilize by filtering
through a 0.22 μm filter.
2. DMEM with 4.5 g/L glucose and without sodium pyruvate
and L-glutamine.
3. 100 mm petri dishes.
168
Benjamin K. Johnson et al.