specific antibodies to cell surface markers, which should be
absent in immature BMDC (e.g., increased expression of
MHC-I). We select the batch concentration, which shows the
best differentiation rate using fluorescence-activated cell sorting (FACS). GM-CSF concentration in the supernatant can
also be determined using an Elisa kit (R&D Systems).
8. Working in RNAse-free conditions: always wear clean lab coat
and gloves. Clean the workbench, the PSM, and all equipment
with RNase AWAY solution prior to the experiment. Use
certified RNAse-free tips, pipets, and Eppendorf tubes.
9. The use of a liquid handling, as Echo Acoustic Liquid
Handling Technology (Labcyte), could be recommended at
this step. This innovative technology allows to transfer smaller
volumes than 250 nL of 10 μM siRNA or compound to have
for example a final concentration of 50 nM in a final volume of
50 μL) and produces faster and more accurate results.
10. The use of an automatic plate washer/dispenser is recommended at this step, to ensure fast, reproducible, and homogenous washing/distribution.
11. We recommended using borosilicate amber glass vials with
hermetic polypropylene PVDF or PTFE-coated screw caps.
Always work in a fume hood, wearing a lab coat and gloves.
As DMSO is very hygroscopic, it is also advised to work under a
light flow of nitrogen to avoid hydration of the solvent.
12. The LysoTracker protocol indicates that “the green LT variant
(DND-26) does not allow pH-sensitive acidification measurements and can only be used to monitor phago-lysosomal fusion
events. The rates of uptake into living cells can occur within
seconds. Unfortunately, these lysosomal probes can exhibit an
‘alkalizing effect’ on the lysosomes, such that longer incubation with this probe can induce an increase in lysosomal
pH. This probe is useful as pH indicator only when it is
incubated with cells for 1–5 min at 37
C.”
13. Bafilomycin is a specific inhibitor of vacuolar H+ ATPase and
inhibits lysosomal acidification, it can be used to block bacterial
escape.
14. The LipidTOX protocol given by the supplier recommends to
dilute 1000Â LipidTOX neutral lipid stain 1:1000 in DPBS to
make a 1Â solution. Routinely, we used LipidTOX neutral lipid
stain 1:2000 diluted, as it is sufficient to stain the cells.
15. You can develop image analysis pipelines by yourself using any
object-oriented computer language (C++, Java, Python, or
similar), but it is advised to use a dedicated software featuring
pre-defined detection algorithms. We used the Columbus system (version 2.3.1; Perkin Elmer) software to develop our own
multi-parameter scripts with the best flexibility.
High-Content Analysis of Host-Pathogen Interactions
699
absent in immature BMDC (e.g., increased expression of
MHC-I). We select the batch concentration, which shows the
best differentiation rate using fluorescence-activated cell sorting (FACS). GM-CSF concentration in the supernatant can
also be determined using an Elisa kit (R&D Systems).
8. Working in RNAse-free conditions: always wear clean lab coat
and gloves. Clean the workbench, the PSM, and all equipment
with RNase AWAY solution prior to the experiment. Use
certified RNAse-free tips, pipets, and Eppendorf tubes.
9. The use of a liquid handling, as Echo Acoustic Liquid
Handling Technology (Labcyte), could be recommended at
this step. This innovative technology allows to transfer smaller
volumes than 250 nL of 10 μM siRNA or compound to have
for example a final concentration of 50 nM in a final volume of
50 μL) and produces faster and more accurate results.
10. The use of an automatic plate washer/dispenser is recommended at this step, to ensure fast, reproducible, and homogenous washing/distribution.
11. We recommended using borosilicate amber glass vials with
hermetic polypropylene PVDF or PTFE-coated screw caps.
Always work in a fume hood, wearing a lab coat and gloves.
As DMSO is very hygroscopic, it is also advised to work under a
light flow of nitrogen to avoid hydration of the solvent.
12. The LysoTracker protocol indicates that “the green LT variant
(DND-26) does not allow pH-sensitive acidification measurements and can only be used to monitor phago-lysosomal fusion
events. The rates of uptake into living cells can occur within
seconds. Unfortunately, these lysosomal probes can exhibit an
‘alkalizing effect’ on the lysosomes, such that longer incubation with this probe can induce an increase in lysosomal
pH. This probe is useful as pH indicator only when it is
incubated with cells for 1–5 min at 37
C.”
13. Bafilomycin is a specific inhibitor of vacuolar H+ ATPase and
inhibits lysosomal acidification, it can be used to block bacterial
escape.
14. The LipidTOX protocol given by the supplier recommends to
dilute 1000Â LipidTOX neutral lipid stain 1:1000 in DPBS to
make a 1Â solution. Routinely, we used LipidTOX neutral lipid
stain 1:2000 diluted, as it is sufficient to stain the cells.
15. You can develop image analysis pipelines by yourself using any
object-oriented computer language (C++, Java, Python, or
similar), but it is advised to use a dedicated software featuring
pre-defined detection algorithms. We used the Columbus system (version 2.3.1; Perkin Elmer) software to develop our own
multi-parameter scripts with the best flexibility.
High-Content Analysis of Host-Pathogen Interactions
699