Intracellular Localization of Brucella abortus and Francisella tularensis in Primary Murine Macrophages - Bacterial Pathogenesis: Methods and Protocols - page 135

beaker. Drain all liquid onto a Kimwipe and immediately invert it on a drop of the
appropriate secondary antibodies. Incubate for 40 min at room temperature.
9. Prepare glass microscopy slides to mount stained coverslips by cleaning them
with ethanol to remove any residue.
10. Wash each coverslip twice in 0.1% saponin-PBS, once in PBS, and finally once
in water to remove salts. Drain all liquid onto a Kimwipe and mount coverslips
on microscopy slides by inverting them (cells facing down) on 10-μL drops of
Mowiol mounting medium. Allow at least2hofpolymerization before viewing
samples on an epifluorescence microscope.
3.4. Phagosomal Integrity Assay (12)
1. This assay is based on the differential labeling of intracellular bacteria depending
on whether they are contained within an intact phagosome or are accessible
to cytoplasmically delivered, specific antibodies due to the disruption of their
phagosome. It relies on a two-step permeabilization of infected cells consisting
of (1) permeabilization of the plasma membrane of live cells using digitonin,
which allows cytoplasmic delivery of antibodies and detection of accessible
bacteria and (2) a complete permeabilization of all eukaryotic membranes using
saponin after PFA fixation, which allows antibody access to all intracellular
bacteria, regardless of their location. The use of different fluorophore-conjugated
secondary antibodies generates a differential labeling of cytoplasmic (double
labeling) and phagosomal bacteria (single labeling), which allows direct analysis
of their intracellular location.
2. Prepare dilutions of mouse anti- F. tularensis LPS (1:2000) and rabbit anti-
calnexin CT (1:250) antibodies in KHM buffer to, respectively, detect cytoplas-
mically accessible bacteria and digitonin-permeabilized cells. Distribute as 30-μL
drops in a humidified immunostaining chamber and preheat to 37 °C.
3. Wash infected BMMs three times with room temperature KHM buffer. Immedi-
ately transfer coverslip(s) in another 24-well plate containing KHM buffer if
necessary.
4. Aspirate KHM buffer and add 500 μL of 50 μg/mL digitonin in KHM buffer
for 1 min at room temperature. Incubation time and digitonin concentration may
vary depending on the cells used, so it is recommended to run preliminary tests.
5. Rinse coverslips in wells with KHM buffer three to four times, carefully
retrieve each coverslip using high-precision tweezers and quickly drain it onto
a Kimwipe.
6. Invert coverslip(s) on pre-heated 30-μL antibody drops and incubate for 10 min
at 37ºC ( see Note 10 ).
7. Put the coverslip(s) back in the 24-well plate and wash three to four times with
PBS.
8. Fix with 2.5% PFA in PBS as described in Subheading 3.3 .
9. Permeabilize cells with permeabilization/blocking buffer for 30 min at room
temperature.