5. Hygromycin: prepare a 1000Â stock solution by dissolving
100 mg of powder in 1 mL deionized water. Filter-sterilize
and store at À20
C.
6. Rifabutin: prepare a 1000Â stock solution by dissolving 10 mg
of powder in 1 mL deionized water. Filter-sterilize and store at
À20
C.
7. Glass beads, 5 mm diameter.
2.3 Preparation
of D. discoideum Cells
for Infection
1. 10 cm cell culture dishes, tissue culture-treated polystyrene.
2. HL5c medium including glucose supplemented with vitamins
and microelements (ForMedium): resuspend 26.55 g of powder in 1 L of deionized water. Filter-sterilize (see Note 1).
3. LoFlo medium (Low fluorescence medium, ForMedium):
resuspend 16.8 g of powder in 1 L of deionized water and
adjust to pH 6.5. Filter-sterilize (see Notes 1 and 3).
2.4 Infection
1. HL5c medium including glucose supplemented with vitamins
and microelements (ForMedium): resuspend 26.55 g of powder in 1 L of deionized water. Filter-sterilize (see Note 1).
2. Blunt needle 25 G Â 3/4
00 ; 0.5 Â 18.
3. 3 mL syringe.
4. Penicillin/streptomycin 100Â stock solution: 10,000 U and
10,000 μg/mL, respectively.
5. Inverted cell culture microscope with a 40Â phase-contrast
objective.
6. Parafilm.
2.5 Single Cell
Experiment
2.5.1 Cleaning
of the InfectChip Device
1. Microfluidic chip (see Subheading 3.5, steps 1–6).
2. 70% ethanol.
3. 100% isopropanol.
4. Double-distilled water.
5. Syringes 5 mL luer lock (BD 309646).
6. 200 mL beaker to store clean and dried device.
7. Bath sonicator.
8. Compressed air can, e.g., Maxxtro, MX-1453.
9. Dry autoclave.
2.5.2 Agarose Sheet
(See Note 4)
1. UltraPure TM Agarose (Invitrogen).
2. HL5c medium including glucose supplemented with vitamins
and microelements (ForMedium): resuspend 26.55 g of powder in 1 L of deionized water. Filter-sterilize (see Note 1).
Monitoring Infection Dynamics of M. marinum in D. Discoideum
187
100 mg of powder in 1 mL deionized water. Filter-sterilize
and store at À20
C.
6. Rifabutin: prepare a 1000Â stock solution by dissolving 10 mg
of powder in 1 mL deionized water. Filter-sterilize and store at
À20
C.
7. Glass beads, 5 mm diameter.
2.3 Preparation
of D. discoideum Cells
for Infection
1. 10 cm cell culture dishes, tissue culture-treated polystyrene.
2. HL5c medium including glucose supplemented with vitamins
and microelements (ForMedium): resuspend 26.55 g of powder in 1 L of deionized water. Filter-sterilize (see Note 1).
3. LoFlo medium (Low fluorescence medium, ForMedium):
resuspend 16.8 g of powder in 1 L of deionized water and
adjust to pH 6.5. Filter-sterilize (see Notes 1 and 3).
2.4 Infection
1. HL5c medium including glucose supplemented with vitamins
and microelements (ForMedium): resuspend 26.55 g of powder in 1 L of deionized water. Filter-sterilize (see Note 1).
2. Blunt needle 25 G Â 3/4
00 ; 0.5 Â 18.
3. 3 mL syringe.
4. Penicillin/streptomycin 100Â stock solution: 10,000 U and
10,000 μg/mL, respectively.
5. Inverted cell culture microscope with a 40Â phase-contrast
objective.
6. Parafilm.
2.5 Single Cell
Experiment
2.5.1 Cleaning
of the InfectChip Device
1. Microfluidic chip (see Subheading 3.5, steps 1–6).
2. 70% ethanol.
3. 100% isopropanol.
4. Double-distilled water.
5. Syringes 5 mL luer lock (BD 309646).
6. 200 mL beaker to store clean and dried device.
7. Bath sonicator.
8. Compressed air can, e.g., Maxxtro, MX-1453.
9. Dry autoclave.
2.5.2 Agarose Sheet
(See Note 4)
1. UltraPure TM Agarose (Invitrogen).
2. HL5c medium including glucose supplemented with vitamins
and microelements (ForMedium): resuspend 26.55 g of powder in 1 L of deionized water. Filter-sterilize (see Note 1).
Monitoring Infection Dynamics of M. marinum in D. Discoideum
187