10. Leave to solidify for 10 min.
11. Detach the agarose sheets from the mold using plastic-tipped
tweezers.
12. Keep them moist by placing them in a small petri dish with
2 mL of HL5c until use.
Inoculate the InfectChip as follows:
13. Resuspend the cells (from Subheading 3.4, step 8) at a density of 3.5 Â 10
6 c/mL in HL5c.
14. Place a micropatterned coverslip in a 35 mm dish.
15. Add 1 mL of the infected cell suspension onto the coverslip.
16. Add 70 mL of HL5c in the beaker.
17. Add penicillin/streptomycin (at a final concentration of 5 μg/
mL streptomycin, 5 U/mL penicillin) to prevent extracellular
growth of M. marinum [24].
18. Aspirate around 60 mL of the media in the syringe, be careful
to avoid bubbles.
19. Prepare the PMMA holder by placing the microfluidic chip in
its center, with the serpentine channel face up, make it adhere
by applying slight pressure on the rim of the chip.
20. Mount the device:
(a) Take gently the coverslip from the dish, maintain horizontally and place it into the aluminum holder.
(b) Add the agarose sheet on top of the inverted microfluidic
chip, on the serpentine channel.
(c) Invert the assembled PDMS chip and membrane and
center them on top of the micropatterned coverslip in
sandwich between the PMMA and the aluminum holder.
(d) Close the device by tightening the screws gently, while
applying pressure on the central part of the chip (see
Note 9).
(e) Connect the syringe to the tubing of the
microfluidic chip.
(f) Gently flow medium (around 2 mL in 5 min) through
the chip to ensure there are no leaks or bubbles.
(g) Prepare a trash beaker with some bleach and parafilm.
21. Go to the spinning disk microscope and use the following
parameters: 63Â glycerol objectives, transmitted light and
laser line 488 nm, temperature controlled at 25
C.
(a) Place the device on the microscope stage, the syringe is
loaded into a syringe pump (WPI, NE-1000), adjust the
flow rate to 10 μL/min.
Monitoring Infection Dynamics of M. marinum in D. Discoideum
191
11. Detach the agarose sheets from the mold using plastic-tipped
tweezers.
12. Keep them moist by placing them in a small petri dish with
2 mL of HL5c until use.
Inoculate the InfectChip as follows:
13. Resuspend the cells (from Subheading 3.4, step 8) at a density of 3.5 Â 10
6 c/mL in HL5c.
14. Place a micropatterned coverslip in a 35 mm dish.
15. Add 1 mL of the infected cell suspension onto the coverslip.
16. Add 70 mL of HL5c in the beaker.
17. Add penicillin/streptomycin (at a final concentration of 5 μg/
mL streptomycin, 5 U/mL penicillin) to prevent extracellular
growth of M. marinum [24].
18. Aspirate around 60 mL of the media in the syringe, be careful
to avoid bubbles.
19. Prepare the PMMA holder by placing the microfluidic chip in
its center, with the serpentine channel face up, make it adhere
by applying slight pressure on the rim of the chip.
20. Mount the device:
(a) Take gently the coverslip from the dish, maintain horizontally and place it into the aluminum holder.
(b) Add the agarose sheet on top of the inverted microfluidic
chip, on the serpentine channel.
(c) Invert the assembled PDMS chip and membrane and
center them on top of the micropatterned coverslip in
sandwich between the PMMA and the aluminum holder.
(d) Close the device by tightening the screws gently, while
applying pressure on the central part of the chip (see
Note 9).
(e) Connect the syringe to the tubing of the
microfluidic chip.
(f) Gently flow medium (around 2 mL in 5 min) through
the chip to ensure there are no leaks or bubbles.
(g) Prepare a trash beaker with some bleach and parafilm.
21. Go to the spinning disk microscope and use the following
parameters: 63Â glycerol objectives, transmitted light and
laser line 488 nm, temperature controlled at 25
C.
(a) Place the device on the microscope stage, the syringe is
loaded into a syringe pump (WPI, NE-1000), adjust the
flow rate to 10 μL/min.
Monitoring Infection Dynamics of M. marinum in D. Discoideum
191