Intracellular Localization of Brucella abortus and Francisella tularensis in Primary Murine Macrophages - Bacterial Pathogenesis: Methods and Protocols - page 131

Fig. 1. Confocal laser scanning micrographs of a C57BL/6 mouse bone marrow-
derived macrophage infected for 24 h with virulent Brucella abortus expressing GFP.
Upper panels: LAMP-1 staining of the late endocytic compartment showing that
replicative BCVs are LAMP-1-negative. Insets show a magnification of a LAMP-1-
negative area (boxed on the whole image), where bacteria replicate (arrow). Lower
panels: Calnexin staining of the endoplasmic reticulum (ER) showing recruitment of
ER markers on the replicative BCVs. Insets show a magnification of calnexin-positive
Brucella -containing vacuoles (BCVs) (arrows). Scale bars, 10 and 2 μm (insets).
cytoplasmic bacteria (phagosomal integrity assay, Fig. 2 ). The successful local-
ization of intracellular bacteria depends on the quality of the immunostaining,
which is achieved through the use of antibodies giving high specific signal-to-
noise ratios. As not all antibodies are useful for immunofluorescence applica-
tions, we provide in this chapter references of commercially available antibodies
that have proven to be of such quality.
3.1. Culture of Bone Marrow-Derived Macrophages
1. To prepare L-929-conditioned medium, grow L-929 mouse fibroblasts in DMEM
supplemented with 10% FBS and 2 m M l-glutamine in tissue-culture flasks at
37 °C under 7% CO 2 with a humidified atmosphere. Expand cells into 175-cm 2
flasks seeded with 2 × 10 6 cells in 80 mL of medium. Grow cells for 7 days,
during which they reach confluency and produce CSF-1, a growth factor required
for bone marrow monocyte differentiation into macrophages and growth. At day
7, collect supernatants, filter them through 0.22 μm membranes, and freeze 10
mL aliquots at -20 °C ( see Note 3 ).