Abstract
Dulbecco and Vogt1 have prepared suspensions of rat liver cells by tryptic digestion in order to produce tissue cultures from single cells. We thought that such suspensions might be suitable for studies of cellular respiration, but found that they contained too few intact cells and many mitochondria. The respiration of mitochondria was found to be unaffected even by concentrations of trypsin strong enough to lyse intact cells. An accidental observation suggested that a technique of mild acid digestion without added enzyme might be effective. A solution similar to Dulbecco and Vogt's phosphate buffer saline1 was used. The phosphates were replaced by the equivalent amount of phosphoric acid and the solution brought to the selected pH value with potassium hydroxide. The most acid solution used was at pH 2.3. This produced the greatest yield of separated cells ; but they barely respired. More alkaline solutions yielded fewer cells, but with increasing alkalinity the respiration-rate rose until with extraction at pH 4 a suspension could be obtained with a respiration-rate of the value given by Krebs2 for liver slices. In practice we thought it best to use a medium of pH 5, which when mixed with the chopped tissue gives a pH value of nearly 6.
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References
Dulbecco, R., and Vogt, M., J. Exp. Med., 99, 167 (1954).
Krebs, H. A., Biochim. Biophys. Acta, 4, 249 (1950).
Hornsey, S., Nature, 176, 744 (1955).
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LONGMUIR, I., AP REES, W. Preparation of Cell Suspensions from Rat Livers. Nature 177, 997 (1956). https://doi.org/10.1038/177997a0
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DOI: https://doi.org/10.1038/177997a0
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