D. discoideum has a density of around 5 Â 10
7 cells/10 cm
dish. To obtain an MOI of 10, the mycobacteria density should
be 5 Â 10
8 bacteria/mL.
2. Centrifuge the mycobacteria at 18,000 Â g for 4 min or
2700 Â g for 10 min.
3. Wash the pellet twice with 1 mL HL5c and resuspend the pellet
in 500 μL of HL5c.
4. Syringe the suspension ten times through a blunt needle to
break up clumps.
5. Aspirate the medium from the D. discoideum culture (see Subheading 3.3) and add 5 mL of HL5c medium without penicillin/streptomycin. Add the bacterial suspension and shake the
dish gently.
6. Seal the 10 cm dish with parafilm and centrifuge twice at
500 Â g for 10 min. Turn the dish by 180
between spins to
redistribute the bacteria suspension.
7. Incubate for 10–20 min to allow phagocytosis.
8. Wash off the extracellular bacteria with 7 mL of HL5c several
times (3–8 repeats). Add and remove the medium gently to
avoid detaching D. discoideum cells from the dish (see Note 8).
After the washes, continue the steps according to the technique you want to use:
– Single cell: see Subheading 3.5,
– High Content: see Subheading 3.6,
– Developmental cycle: see Subheading 3.7.
3.5 Single Cell
Experiment
Prepare the InfectChip device as follows:
1. Before and after each experiment, fill the microfluidic chip with
70% ethanol and immerse for at least 24 h.
2. Replace 70% ethanol by 100% isopropanol and sonicate for 1 h
at 4
C in a bath sonicator.
3. Clean tubing and device by flowing 10 mL of 70% ethanol and
then rinse by flowing 20 mL of doubled-distilled water.
4. Dry using compressed air from a can (Maxxtro, MX-1453).
5. Autoclave at 121
C for 20 min.
6. Cool the chip to RT before use.
Prepare the agarose sheet as follows:
7. Prepare a 2% agarose sheet. Dissolve 0.2 g of agarose in 10 mL
of HL5c by heating in a microwave.
8. Pour solution onto a mold to make 25 mm diameter and
200 μm high agarose sheets.
9. Place a glass wafer on the filled mold and place a full 1 L bottle
on top to apply pressure.
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Manon Mottet et al.
7 cells/10 cm
dish. To obtain an MOI of 10, the mycobacteria density should
be 5 Â 10
8 bacteria/mL.
2. Centrifuge the mycobacteria at 18,000 Â g for 4 min or
2700 Â g for 10 min.
3. Wash the pellet twice with 1 mL HL5c and resuspend the pellet
in 500 μL of HL5c.
4. Syringe the suspension ten times through a blunt needle to
break up clumps.
5. Aspirate the medium from the D. discoideum culture (see Subheading 3.3) and add 5 mL of HL5c medium without penicillin/streptomycin. Add the bacterial suspension and shake the
dish gently.
6. Seal the 10 cm dish with parafilm and centrifuge twice at
500 Â g for 10 min. Turn the dish by 180
between spins to
redistribute the bacteria suspension.
7. Incubate for 10–20 min to allow phagocytosis.
8. Wash off the extracellular bacteria with 7 mL of HL5c several
times (3–8 repeats). Add and remove the medium gently to
avoid detaching D. discoideum cells from the dish (see Note 8).
After the washes, continue the steps according to the technique you want to use:
– Single cell: see Subheading 3.5,
– High Content: see Subheading 3.6,
– Developmental cycle: see Subheading 3.7.
3.5 Single Cell
Experiment
Prepare the InfectChip device as follows:
1. Before and after each experiment, fill the microfluidic chip with
70% ethanol and immerse for at least 24 h.
2. Replace 70% ethanol by 100% isopropanol and sonicate for 1 h
at 4
C in a bath sonicator.
3. Clean tubing and device by flowing 10 mL of 70% ethanol and
then rinse by flowing 20 mL of doubled-distilled water.
4. Dry using compressed air from a can (Maxxtro, MX-1453).
5. Autoclave at 121
C for 20 min.
6. Cool the chip to RT before use.
Prepare the agarose sheet as follows:
7. Prepare a 2% agarose sheet. Dissolve 0.2 g of agarose in 10 mL
of HL5c by heating in a microwave.
8. Pour solution onto a mold to make 25 mm diameter and
200 μm high agarose sheets.
9. Place a glass wafer on the filled mold and place a full 1 L bottle
on top to apply pressure.
190
Manon Mottet et al.