8. Control the efficiency of the washing step by checking the
presence of extracellular bacteria with an inverted cell culture
microscope.
9. When closing the device with the screws, the pressure applied
on the central portion of the chip should be equally distributed.
This procedure prevents the D. discoideum cells from flowing
out of the chambers.
10. Autofocusing, which consists in the automatic selection of the
most contrasted plane in a Z-stack, is available with the
spinning disk microscope described here. This is especially
important for long-term imaging, a significant shift in the
Z axis over time could lead to the loss of the focal plane.
11. This condition is needed to test the efficacy of bacterial growth
inhibition by antibiotics.
12. Cells were sorted on an SH-800 device (Sony) using a 100 μm
sorting chip at 4
C by cooling both the input tube holder and
the collection tube rack. The gates for GFP+ and mCherry+
fractions were conservative in order to prevent cross contamination. On average, 2500 events/second were acquired in
order to keep a low differential pressure and improve the
resolution quality. Note that for good sorting efficiency, the
original suspension should contain 30–50% of infected cells.
13. The dynamics of the aggregation phase of D. discoideum are
sensitive to cell number, temperature, vibration, phototoxicity,
and humidity. To increase the efficiency and speed of the
aggregation phase, a higher density of cells can be used. Since
the aggregation depends on chemotaxis, a low volume of
buffer will facilitate aggregation; however, cell drying must
be avoided. A temperature of 22
C instead of 25
C is used
to maintain infected and FAC-sorted D. discoideum health
during the developmental cycle and allow monitoring of collective behavior feasible.
14. Infected cells have weaker substrate attachment and are thus
prone to be washed away when exchanging medium for low
nutrient buffer. If the FAC-sorting is done late during the 6-h
incubation, alternatively plate the cells in the ibidi dish and
change the medium to SorMC in one step, therefore avoiding
the washing step within the well. However, it is important to
stay consistent within one experiment.
15. The meniscus effect in the well can cause inhomogeneity in cell
plating as well as imaging and illumination distortions. To
ensure a homogeneous distribution of cells in the well of the
8-well ibidi dish, use a volume of 400 μL of HL5c medium.
Once the cells are in SorMC (circa 100 μL, as described above),
the meniscus effect is stronger, due to the lower volume, and
thus the aggregation phase is typically sped up in the center of
the well.
Monitoring Infection Dynamics of M. marinum in D. Discoideum
199
presence of extracellular bacteria with an inverted cell culture
microscope.
9. When closing the device with the screws, the pressure applied
on the central portion of the chip should be equally distributed.
This procedure prevents the D. discoideum cells from flowing
out of the chambers.
10. Autofocusing, which consists in the automatic selection of the
most contrasted plane in a Z-stack, is available with the
spinning disk microscope described here. This is especially
important for long-term imaging, a significant shift in the
Z axis over time could lead to the loss of the focal plane.
11. This condition is needed to test the efficacy of bacterial growth
inhibition by antibiotics.
12. Cells were sorted on an SH-800 device (Sony) using a 100 μm
sorting chip at 4
C by cooling both the input tube holder and
the collection tube rack. The gates for GFP+ and mCherry+
fractions were conservative in order to prevent cross contamination. On average, 2500 events/second were acquired in
order to keep a low differential pressure and improve the
resolution quality. Note that for good sorting efficiency, the
original suspension should contain 30–50% of infected cells.
13. The dynamics of the aggregation phase of D. discoideum are
sensitive to cell number, temperature, vibration, phototoxicity,
and humidity. To increase the efficiency and speed of the
aggregation phase, a higher density of cells can be used. Since
the aggregation depends on chemotaxis, a low volume of
buffer will facilitate aggregation; however, cell drying must
be avoided. A temperature of 22
C instead of 25
C is used
to maintain infected and FAC-sorted D. discoideum health
during the developmental cycle and allow monitoring of collective behavior feasible.
14. Infected cells have weaker substrate attachment and are thus
prone to be washed away when exchanging medium for low
nutrient buffer. If the FAC-sorting is done late during the 6-h
incubation, alternatively plate the cells in the ibidi dish and
change the medium to SorMC in one step, therefore avoiding
the washing step within the well. However, it is important to
stay consistent within one experiment.
15. The meniscus effect in the well can cause inhomogeneity in cell
plating as well as imaging and illumination distortions. To
ensure a homogeneous distribution of cells in the well of the
8-well ibidi dish, use a volume of 400 μL of HL5c medium.
Once the cells are in SorMC (circa 100 μL, as described above),
the meniscus effect is stronger, due to the lower volume, and
thus the aggregation phase is typically sped up in the center of
the well.
Monitoring Infection Dynamics of M. marinum in D. Discoideum
199