5. As a control, use:
(a) Uninfected D. discoideum cells resuspended also at
1.3 Â 10
6 cells/mL: 12 μL, fill up to 200 μL with filtered
HL5c with 5 μg/mL streptomycin and 5 U/mL
penicillin.
(b) Infected
D. discoideum resuspended
also at
1.3 Â 10
6 cells/mL: 12 μL, fill volume to 200 μL with
filtered HL5c with 5 μg/mL streptomycin and 5 U/mL
penicillin and rifabutin 10 μg/mL.
(c) 1 μL of syringed bacteria, fill up to 200 μL with 7H9.
(d) 1 μL of syringed bacteria, fill up to 200 μL with 7H9 with
5 μg/mL streptomycin and 5 U/mL penicillin (see Note
11).
6. Seal the 96-well plate with permeable gas films.
7. Image cells every hour by IXM Confocal up to 72 h, using a
40Â objective, collecting 9–16 fields per well, depending on
the number of conditions.
3.7 Developmental
Cycle
1. Perform the infection protocol as described (Subheading 3.4)
with GFP-LifeAct-expressing D. discoideum and the mCherryexpressing M. marinum strain.
2. After washing (see Subheading 3.4, step 8), collect infected
cells, centrifuge at 500 Â g for 5 min, discard supernatant,
and resuspend cells in a volume of 1–3 mL of HL5c (volume
depending on exact conditions during FAC sorting).
3. Incubate the infection for 6 h at 25
C before switching to low
nutrient conditions, which triggers the developmental cycle.
4. Cell sorting is performed during the 6 h incubation time by
following the steps below:
(a) Select the cell population of interest by gating on cell
diameter (forward-scatter) and granularity (side-scatter).
(b) Select a subpopulation of infected cells based on the signal
intensity (mCherry and FITC channels) (see Note 12).
(c) Collect infected cells in 15 mL tubes filled with 3 mL
HL5c; for good sorting efficiency, the suspension should
contain around 50% of infected cells.
(d) Before plating the cells, spin them down (5 min, 500 Â g)
and resuspend in 400 μL HL5c.
5. Plate at least 5 Â 10
5 cells in a volume of 400 μL HL5c per well
of the 8-well ibidi dish (see Notes 13 and 14).
6. After 6 h of infection, switch to low nutrient conditions by
gently exchanging medium for SorMC, including at least one
rinsing step, and leaving each well with a buffer overlay of circa
100 μL (see Notes 14 and 15).
Monitoring Infection Dynamics of M. marinum in D. Discoideum
193
(a) Uninfected D. discoideum cells resuspended also at
1.3 Â 10
6 cells/mL: 12 μL, fill up to 200 μL with filtered
HL5c with 5 μg/mL streptomycin and 5 U/mL
penicillin.
(b) Infected
D. discoideum resuspended
also at
1.3 Â 10
6 cells/mL: 12 μL, fill volume to 200 μL with
filtered HL5c with 5 μg/mL streptomycin and 5 U/mL
penicillin and rifabutin 10 μg/mL.
(c) 1 μL of syringed bacteria, fill up to 200 μL with 7H9.
(d) 1 μL of syringed bacteria, fill up to 200 μL with 7H9 with
5 μg/mL streptomycin and 5 U/mL penicillin (see Note
11).
6. Seal the 96-well plate with permeable gas films.
7. Image cells every hour by IXM Confocal up to 72 h, using a
40Â objective, collecting 9–16 fields per well, depending on
the number of conditions.
3.7 Developmental
Cycle
1. Perform the infection protocol as described (Subheading 3.4)
with GFP-LifeAct-expressing D. discoideum and the mCherryexpressing M. marinum strain.
2. After washing (see Subheading 3.4, step 8), collect infected
cells, centrifuge at 500 Â g for 5 min, discard supernatant,
and resuspend cells in a volume of 1–3 mL of HL5c (volume
depending on exact conditions during FAC sorting).
3. Incubate the infection for 6 h at 25
C before switching to low
nutrient conditions, which triggers the developmental cycle.
4. Cell sorting is performed during the 6 h incubation time by
following the steps below:
(a) Select the cell population of interest by gating on cell
diameter (forward-scatter) and granularity (side-scatter).
(b) Select a subpopulation of infected cells based on the signal
intensity (mCherry and FITC channels) (see Note 12).
(c) Collect infected cells in 15 mL tubes filled with 3 mL
HL5c; for good sorting efficiency, the suspension should
contain around 50% of infected cells.
(d) Before plating the cells, spin them down (5 min, 500 Â g)
and resuspend in 400 μL HL5c.
5. Plate at least 5 Â 10
5 cells in a volume of 400 μL HL5c per well
of the 8-well ibidi dish (see Notes 13 and 14).
6. After 6 h of infection, switch to low nutrient conditions by
gently exchanging medium for SorMC, including at least one
rinsing step, and leaving each well with a buffer overlay of circa
100 μL (see Notes 14 and 15).
Monitoring Infection Dynamics of M. marinum in D. Discoideum
193