Intracellular Localization of Brucella abortus and Francisella tularensis in Primary Murine Macrophages - Bacterial Pathogenesis: Methods and Protocols - page 137

flattened and contained many translucent vacuoles, they are probably activated
by residual endotoxins or other activating molecules in the FBS or the L-929-
conditionedmedium. In this case, the percentage of L-929-conditonedmediumcan
be decreased and/or reagents with lower endotoxin levels should be used.
5. Theoretical MOIs refer to the number of bacteria added to the wells containing
the macrophages. Because the synchronized infection procedure ( see point 3
of Subheading 3.2 ) allows bacterial uptake for a limited time, only a fraction
of the bacteria added to the well is phagocytosed. Consequently, the actual
MOI (number of bacteria internalized by macrophages) is lower and has to be
estimated by microscopy or counts of viable intracellular bacteria following
gentamicin treatment.
6. A rapid warm up of the chilled, infected BMMs is essential to induce an efficient
uptake of the bacteria in close contact to the BMMs and synchronize their
subsequent intracellular trafficking.
7. Shorter fixation times with PFA, such as 10 min, are possible, as they sufficiently
fix intracellular structures and can help preserve sensitive epitopes. However,
they might not be sufficient to kill all bacteria in the samples, which is a concern
for highly infectious (Biosafety Level 3) pathogens.
8. Triton X-100 permeabilization can help extract epitopes from intracellular
membranes or cytoskeletal structures and yield to better staining. Because the
Triton X-100 effect on cell membranes is irreversible, it is not required to keep
it in the antibody dilutions buffers.
9. Optimal dilutions and incubation times that generate strong labeling and specific
detection of the epitope have to be determined for each antibody. It is essential
to the quality of the immunostaining to drain all liquid from the coverslip before
incubation with primary and secondary antibodies, as remaining liquid will dilute
the applied antibodies further.
10. The short incubation time (10 min) required at this stage relates to the relative
fragility of digitonin-permeabilized primary macrophages. Other cells might be
more resistant to digitonin treatment and allow for a longer incubation with
antibodies. It is nonetheless important to pre-establish and use antibody dilutions
that ensure detectable signals for both bacteria and calnexin.
Acknowledgments
We are grateful to Leigh Knodler for critical reading of the manuscript. This
work was supported by the Intramural Research Program of the NIH, National
Institute of Allergy and Infectious Diseases.
References
1. Knodler, L. A., Celli, J., and Finlay, B. B. (2001) Pathogenic trickery: deception
of host cell processes. Nat. Rev. Mol. Cell Biol . 2 , 578-588.
2. Celli, J. (2005) Surviving inside a macrophage: the many ways of Brucella . Res.
Microbiol . 157 , 93-98.