Download Isolated Hepatocytes Preparation, Properties and by R.H. Burdon and P.H. van Knippenberg (Eds.) PDF
By R.H. Burdon and P.H. van Knippenberg (Eds.)
This booklet units out essentially and successfully the guidance and dealing equipment of laboratory concepts concerning remoted hepatocytes and may make existence more straightforward for each laboratory employee interested in those strategies.
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Extra info for Isolated Hepatocytes Preparation, Properties and Applications
Example text
3. 1. Animals The description given here applies to the preparation of hepatocytes from adult rats in either the fed or fasted state. In general, rats of Ch. 2. HIGH-YIELD PREPARATION 17 150-300 g body weight are the most convenient from a surgical point of view; the liver weights of such animals range between 5 and 10 g. For reliable results it is desirable to maintain consistency in regard to the strain, sex and weight of the rat. Of equal importance is maintenance of a regular feeding schedule, and a consistent light and dark cycle.
2. HIGH-YIELD PREPARATION 39 found associated with hepatocytes when either Sigma Type VII or Worthington type CISPA collagenase from CI. histolyticum, each of which contain only small amounts of contaminating proteases, were used. However, cell yields were only 3 x lo7 intact cells compared with the expected 25-50 x 107 cells for livers of comparable size digested with crude collagenase. Experience indicates that the only reasonably sure way to secure an effective batch of collagenase is to obtain three or four small samples from the supplier of choice and test them.
For these reasons the use of Krebs-Henseleit bicarbonate-saline is recommended. g. 100 mM HEPES) is more convenient than the use of bicarbonate, which, though a better buffer, requires a gas phase of CO,, to allow the correct pH to be maintained. Moreover, Seglen (1973, 1976) has simplified the perfusion on the basis that, in his view, continuous oxygenation of the perfusing medium is not required. Under these circumstances, the employment of HEPES is obviously advantageous. , 1989). We have conducted a number of studies in which cells from fasted animals were prepared according to Seglen’s recommendations.