Intracellular Localization of Brucella abortus and Francisella tularensis in Primary Murine Macrophages - Bacterial Pathogenesis: Methods and Protocols - page 132

Fig. 2. Confocal laser scanning micrographs of a C57BL/6 mouse bone marrow-
derived macrophage infected for 24 h with Francisella tularensis and subjected to
the phagosomal integrity assay. Left-hand panel: Calnexin staining of the endoplasmic
reticulum (ER) following digitonin treatment that demonstrates permeabilization of the
cell; middle panel: Francisella staining following digitonin treatment that demonstrates
that replicating Francisella are detectable using cytoplasmically-delivered antibodies;
and right-hand panel: Francisella staining following complete permeabilization with
saponin that detect all intracellular bacteria. The arrowhead indicates the only phago-
somal bacterium, which is not detected following cytoplasmic delivery of anti-
Francisella antibodies (middle panel). Scale bar, 10 μm.
2. Euthanize mice using a lethal dose of isofluorane and immediately remove femurs
from hind legs using sterile dissection tools. It is important to remove and
discard all the tissue around the bones to avoid further contamination of the
macrophage culture with fibroblasts. Femurs can be kept in cold DMEM until
further processing (not more than1hispreferable).
3. In a biosafety cabinet, prepare a six-well plate with one well containing 70%
ethanol and four others 2 mL of DMEM. Surface sterilize each femur by rapidly
passing it in 70% ethanol, rinse it with DMEM in the next well, and then transfer
it in the third well. Scrape off any remaining tissue around the bone, transfer the
clean bone into the next well and cut both ends with a scalpel.
4. Transfer immediately to the next well and flush cells out gently with a 1-mL
syringe fitted with a 25G 5/8 -gauge needle, by passing DMEM 3-4 times through
the bone from each side until the bone is white. Be careful not to scrape the
bone with the needle, as this will release osteoclasts. Avoid bubbles as much as
possible. Collect cells in a sterile tube and put on ice. You should obtain 1-2 × 10 7
monocytes/femur.
5. Put the cell suspension (from 1.5-2 femurs) in culture in a 150 mm non-tissue
culture-treated dish in 30 mL of DMEM supplemented with 10% FBS, 2 m M
l-glutamine, and 10% L-929-conditioned medium for 5 days at 37 °C under 7%
CO 2 with a humidified atmosphere. Monocytes will progressively differentiate
into macrophages (BMMs), which will adhere loosely to the dish.
6. On day 5, rock dishes gently back and forth to dislodge non-adherent cells (non-
differentiated monocytes). Wash twice with 15 mL of D-PBS containing MgCl 2