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note
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The solubility of gas is temperature dependent, as is the partial pressure due to water vapor. In this report, the following assumptions ale made regarding oxygen solubility. At 37°, the partial pressure of water vapor is ∼45 mm of Hg; thus, atmospheric pressure of 760 mm of Hg of air with saturated water vapor allows for oxygen (21%) + nitrogen (79%) partial pressures of 149 + 566 mm of Hg, respectively. The solubility of oxygen in physiological saline (or equivalent) equilibrated with water vapor-saturated air at 37° is about 0.20 mM. At 25° and 4°, the corresponding solubility is about 0.25 and 0.45 mM, respectively. In the text, we report absolute concentrations (in micromolar) and mm of Hg or percentage oxygen values assuming a temperature of 37°.
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31
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Polycarbonate is the most oxygen permeable of plastics made for tissue culture use (dishes are made by NUNC). The least toxic and water-impermeable tubing is made from silicone, but it is by far the worst in the sense of high oxygen permeability. It is not the present intention to make a blanket condemnation of experiments using plastics. Rather, it should be appreciated that many oxygen-dependent chemical and biological phenomena are very sensitive to the range of oxygen below 2% (some much lower still). Thus, use of plastics may prevent a proper understanding of the oxygen dependence of various biological phenomena.
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To promote cell attachment, glass is cleaned, heated to 500°, and then conditioned by overnight treatment with water containing 50 mM sodium carbonate and 15% serum, followed by 1 hr with water containing tissue culture grade gelatin (0.1%). The gelatin is removed, but the thin remaining film is allowed to dry onto the glass under UV light in a sterile hood.
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43
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85030746647
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note
-
It is important to realize that the internal volume of gas regulators is several tens of milliliters. Thus, when a regulator is first connected to a gas cylinder (or pressurized after disuse), this internal oxygen-containing volume is highly pressurized by the cylinder gas (e.g., nitrogen) and can cause a long-lasting contamination of oxygen if used at low flow rates. To prevent this, the regulator must be purged by a series of on-off cycles of the high-pressure cylinder valve, each followed by release of the gas from the regulator.
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