9. 3-stop pump tubing and input flow path tubing (Masterflex L/
S-14 platinum-cured silicone tubing, internal diameter
(ID) 0.063 in) (Cole-Parmer Instrument Co).
10. Output flow path tubing (platinum-cured silicone tubing, ID
0.125 in) (Cole-Parmer Instrument Co).
11. Barbed Luer fittings (Cole-Parmer Instrument Co).
12. Barbed connectors (Cole-Parmer Instrument Co).
13. Tubing clamps (Cole-Parmer Instrument Co).
14. Syringe filter, 0.2μm pore size, 13 mm diameter.
15. Stir plate.
16. Lead collar.
17. Upright incubator with glass door or window.
18. 70% ethanol.
19. 1, 3, 5 and 10 mL Luer-lock syringes.
20. Blunt-end needles (16 Â 1.5 in.) e.g. from StemCell
Technologies.
21. 0.5% H 2 O 2 disinfectant wipes.
22. Sterile 70% alcohol prep pads.
23. Nemco condiment chiller (WebstaurantStore).
24. Microclaves (McKesson Medical-Surgical).
25. New Era syringe pump (Scientific Instrument Services).
26. Solenoid pinch valves (Bio-Chem Fluidics).
27. PC-16 programmable valve controller (Bioscience Tools).
3 Methods
3.1 System
Assembly, Sterilization
and Setup
Components of the system are depicted schematically in Fig. 1. An
image of the complete experimental setup is shown in Fig. 2.
The reservoir cap on the 4 L medium supply tank has a silicone
rubber insert with two L-shaped stainless-steel tubes running
through it, with their longer ends oriented toward the inside of
the tank (see Fig. 1 inset and Note 1). One tube acts as a vent, while
the other is used as a siphon.
1. Connect a 0.2μm filter (13 mm) with Luer fitting to the
vent tube.
2. Connect a length of autoclavable silicone tubing to the siphon
tube in order to pump medium from the bottom of the
reservoir.
3. Using Luer fittings, connect Masterflex L/S-14 (0.063 in ID)
tubing (or its equivalent) from the external end of the siphon
718
A. Brett Mason and Ve ´ ronique Dartois
S-14 platinum-cured silicone tubing, internal diameter
(ID) 0.063 in) (Cole-Parmer Instrument Co).
10. Output flow path tubing (platinum-cured silicone tubing, ID
0.125 in) (Cole-Parmer Instrument Co).
11. Barbed Luer fittings (Cole-Parmer Instrument Co).
12. Barbed connectors (Cole-Parmer Instrument Co).
13. Tubing clamps (Cole-Parmer Instrument Co).
14. Syringe filter, 0.2μm pore size, 13 mm diameter.
15. Stir plate.
16. Lead collar.
17. Upright incubator with glass door or window.
18. 70% ethanol.
19. 1, 3, 5 and 10 mL Luer-lock syringes.
20. Blunt-end needles (16 Â 1.5 in.) e.g. from StemCell
Technologies.
21. 0.5% H 2 O 2 disinfectant wipes.
22. Sterile 70% alcohol prep pads.
23. Nemco condiment chiller (WebstaurantStore).
24. Microclaves (McKesson Medical-Surgical).
25. New Era syringe pump (Scientific Instrument Services).
26. Solenoid pinch valves (Bio-Chem Fluidics).
27. PC-16 programmable valve controller (Bioscience Tools).
3 Methods
3.1 System
Assembly, Sterilization
and Setup
Components of the system are depicted schematically in Fig. 1. An
image of the complete experimental setup is shown in Fig. 2.
The reservoir cap on the 4 L medium supply tank has a silicone
rubber insert with two L-shaped stainless-steel tubes running
through it, with their longer ends oriented toward the inside of
the tank (see Fig. 1 inset and Note 1). One tube acts as a vent, while
the other is used as a siphon.
1. Connect a 0.2μm filter (13 mm) with Luer fitting to the
vent tube.
2. Connect a length of autoclavable silicone tubing to the siphon
tube in order to pump medium from the bottom of the
reservoir.
3. Using Luer fittings, connect Masterflex L/S-14 (0.063 in ID)
tubing (or its equivalent) from the external end of the siphon
718
A. Brett Mason and Ve ´ ronique Dartois