4. 100 μm microfluidic sorting chip (CoreFinder™): This sorting
chip contains microfluidic channels for controlling the flow of
sheath fluid and sample. The sample is interrogated within the
chip before passing through the integrated orifice to be sorted.
2.8 Analysis
and Quantification
1. ImageJ software (https://imagej.net/Fiji/Downloads).
2. SlideBook 3i software.
3. MetaXpress
® Molecular device software.
4. Excel
®
Microsoft
® Office
® software.
5. Handbrake (https://handbrake.fr/).
6. Imaris
® by Bitplane.
3 Methods
3.1 Cell Culture
1. Recover D. discoideum cells from frozen spore stocks stored in
liquid nitrogen (see Note 6).
2. Culture D. discoideum Ax2(Ka) or Ax2-214 in adherent culture
in HL5c medium containing penicillin/streptomycin (100 U
and 100 μg/mL, respectively) at 22
C to a maximum density
of 5 Â 10
7 cells/10 cm dish.
3. Culture D. discoideum Ax2(Ka)-GFP-LifeAct strain in HL5c
medium containing penicillin/streptomycin (100 U and
100 μg/mL, respectively) and 50 μg/mL of hygromycin.
3.2 Mycobacterial
Culture
1. Recover M. marinum (see Note 2) from À80
C glycerol stocks
and inoculate into 5 mL of 7H9 medium (plus appropriate
antibiotics depending on the bacterial strain, mCherryexpressing
variant
at
100
μg/mL
hygromycin,
GFP-expressing variant at 50 μg/mL kanamycin).
2. Grow with shaking at 150 rpm at 32
C.
3. Inoculate 25 mL of 7H9 medium to ~0.2 Â 10
8 bacteria/mL
in flasks containing 5 mm glass beads to minimize clumping.
4. Cultivate with shaking until OD 600 ¼ 1 after ~24 h,
corresponding to about 1.8 Â 10
8 bacteria/mL.
3.3 Preparation
of Cells for Infection
1. Plate D. discoideum cells in 10 cm dishes at 1 Â 10
7 cells
(around 40% of confluency), in HL5c medium without penicillin/streptomycin.
2. Incubate for 24 h at 22
C. The plates should be approximately
at 4 Â 10
7 cells (around 90–95% of confluency) on the day of
infection.
3.4 Infection
1. Measure the OD 600 (see Note 7) of the mycobacterial culture
and calculate the volume necessary for a multiplicity of infection (MOI) of 10. At the optimal confluency for infection,
Monitoring Infection Dynamics of M. marinum in D. Discoideum
189
chip contains microfluidic channels for controlling the flow of
sheath fluid and sample. The sample is interrogated within the
chip before passing through the integrated orifice to be sorted.
2.8 Analysis
and Quantification
1. ImageJ software (https://imagej.net/Fiji/Downloads).
2. SlideBook 3i software.
3. MetaXpress
® Molecular device software.
4. Excel
®
Microsoft
® Office
® software.
5. Handbrake (https://handbrake.fr/).
6. Imaris
® by Bitplane.
3 Methods
3.1 Cell Culture
1. Recover D. discoideum cells from frozen spore stocks stored in
liquid nitrogen (see Note 6).
2. Culture D. discoideum Ax2(Ka) or Ax2-214 in adherent culture
in HL5c medium containing penicillin/streptomycin (100 U
and 100 μg/mL, respectively) at 22
C to a maximum density
of 5 Â 10
7 cells/10 cm dish.
3. Culture D. discoideum Ax2(Ka)-GFP-LifeAct strain in HL5c
medium containing penicillin/streptomycin (100 U and
100 μg/mL, respectively) and 50 μg/mL of hygromycin.
3.2 Mycobacterial
Culture
1. Recover M. marinum (see Note 2) from À80
C glycerol stocks
and inoculate into 5 mL of 7H9 medium (plus appropriate
antibiotics depending on the bacterial strain, mCherryexpressing
variant
at
100
μg/mL
hygromycin,
GFP-expressing variant at 50 μg/mL kanamycin).
2. Grow with shaking at 150 rpm at 32
C.
3. Inoculate 25 mL of 7H9 medium to ~0.2 Â 10
8 bacteria/mL
in flasks containing 5 mm glass beads to minimize clumping.
4. Cultivate with shaking until OD 600 ¼ 1 after ~24 h,
corresponding to about 1.8 Â 10
8 bacteria/mL.
3.3 Preparation
of Cells for Infection
1. Plate D. discoideum cells in 10 cm dishes at 1 Â 10
7 cells
(around 40% of confluency), in HL5c medium without penicillin/streptomycin.
2. Incubate for 24 h at 22
C. The plates should be approximately
at 4 Â 10
7 cells (around 90–95% of confluency) on the day of
infection.
3.4 Infection
1. Measure the OD 600 (see Note 7) of the mycobacterial culture
and calculate the volume necessary for a multiplicity of infection (MOI) of 10. At the optimal confluency for infection,
Monitoring Infection Dynamics of M. marinum in D. Discoideum
189