Prevention of radioactive contamination of cell-culture incubators during metabolic labeling experiments.

Labeling of eukaryotic cells with radioactive nutrients is a popular method in the detection of biomacromolecules. Less popular and much ignored is the radioactive contamination of cell-culture equipment that usually accompanies this activity. The reason for this contamination is the production of radioactive vapors or gases, either by spontaneous chemical decomposition or through metabolic conversion by the cultured cell. Examples are volatile tritiated products, including tritiated water, produced during metabolic labeling with 3H-leucine and volatile sulfur compounds, such as H2S and CH3SH, produced during labeling with 35S-methionine or 35S-cysteine (2). The problem has become more pronounced through the introduction of relatively inexpensive bacterially produced 35S-methionine/35S-cysteine in vivo labeling mixtures, which contain considerable amounts of volatile impurities. Although many laboratories designate one incubator for cell-labeling purposes in an effort to contain radioactive contamination, significant amounts of radioactivity can easily spread. One solution to reduce the amount of radioactive air contamination is the use of activated charcoal, which can bind many volatile compounds (2). However, even in the presence of activated charcoal, the inside walls and shelves of the incubator can still collect considerable amounts of radioactivity (as measured by wipe tests) and thus remain a potential source of radioactive spread. In addition, certain volatile compounds, such as tritiated water, do not specifically bind to activated charcoal. Another solution would be to carry out the labeling in an air-tight container. For this purpose, it appeared that the Nalgene transparent polycarbonate desiccator (Catalog No. 5311; Nalge, Rochester, NY, USA) is very suitable. This container is sufficiently large (11 L) for space and oxygen requirements, but small enough (33-cm high, 28-cm in diameter) to fit into a cell-culture or general-purpose incubator. Moreover, the top can be tightly clamped to the bottom part of the desiccator with four large office binder clips (5-cm wide, 2cm opening); a silicone ring between the two parts ensures a leak-proof seal (Figure 1). A level surface required for cell culturing is created by a round 230mm plate that is inserted into the chamber. This can be any standard 230-mm desiccator plate (e.g., plastic: Nalgene Catalog No. 5312-0230; Nalge) or, as shown, a custom-made, 7-mm-thick punctured Perspex plate, with 3 notches to facilitate removal. To allow flushing of the chamber with an appropriate gas mixture, two stopcocks (Nalgene Catalog No. 71-5311-0010; one comes with desiccator) were glued into preexisting holes in the top and bottom parts of the desiccator using an all-purpose glue (Seal-all; Allen Products, Detroit, MI, USA). Flushing (Figure 1) can conveniently be carried out in a fume-hood, further minimizing radioactive contamination. An atmosphere suitable for culturing cells in Dulbecco’s modified Eagle medium (DMEM)-based culture medium, which is most widely used in metabolic labeling experiments, can be established by flushing the chamber with a mixture of 10% CO2, 20% O2 and 70% N2. This mixture is available from most suppliers of air products. Flushing the container with 5 vol of this gas takes less than 2 min at moderately low gas flow (30 L/min). Gas flow is easily measured by timed inflation of a plastic bag of known volume. Closing of the stopcocks and positioning of the clamps create a tightly closed container that can be safely transported to an incubator. CAUTION: It is advisable to position the four clamps after flushing of the