Shay/Wright Lab Low Oxygen Tissue Culture Chambers
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•
Cheap, provides an easy way to devote part of an incubator to low oxygen conditions. Modular,
meaning there is no cross-contamination between culture infections in the incubator, and if the
CO2 in the incubators fails, the cells in the gas-tight low oxygen chambers are fine.
•
Chambers are Nalgene One Liter (32 Oz) polymethylpentene Straight-Side Wide-Mouth Jars
stood on their heads with two holes drilled in the “bottoms” (which are now the tops). The holes
(1/2 inch, drilled with a home drill-press using a flat ½” wood drill bit) get sealed with silicone
rubber stoppers. The lid from a 10 cm petri dish serves to create a flat surface on which TC
plates or flasks rest.
•
Put in the flasks, screw the container closed, flush for 2 minutes at 2 psi with a tri-gas mix (e.g.,
2% oxygen, 7% CO2, 91% nitrogen), then plug the holes with the silicone stoppers.
•
Note that a T25 contains 75 ml gas space and a 10 cm dish contains 100 ml of gas space. The
one liter jar actually contains 1.2 L of gas space. A stack of four 10 cm dishes thus contains 1/3
the total gas space, so that even if all of the exterior volume initially contains 2% oxygen, after it
equilibrates the oxygen will be (0.4L*21% + 0.8L*2%/1.2L = ) 8% oxygen final. Thus you
shouldn’t put in too many dishes, or should re-gas it after an hour to drive the oxygen lower if
you really need it very low (tissue O2 is 1%-6%). The reason for using 7% rather than 5% CO2
is to compensate for the lack of CO2 inside the dishes to begin with.
• Test the system by gassing and then just putting it on a bench for a week. If they leak, the CO2
will leak out and the medium will turn alkaline.
Nalgene 2117-1000 polymethylpentene
1 liter Wide-Mouth Jar
10 cm petri dish cover inverted on
top of “bump” on undersurface of lid
Two ½ inch diameter
holes drilled in top
Threads coated with silicone vacuum
grease so the lid screws shut easily
and forms gas-tight seal
Size “15D” silicone rubber stoppers
(15mm diameter at big end)
Bubble tubing connected to low oxygen tank
Gauge to step down
delivery pressure to 2 psi
We use 2% Oxygen, 7% CO2, 91% nitrogen
Of the different configurations we have tried (see
below), the Nalgene 1L containers are by far the most
gas-tight. The others work for 24-well plates or 15 cm
dishes, but they leak and need to be regassed
twice/week.