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Counting cells as DNA: Estimation of ovine pituitary cells on Sephadex columns by Mary G. Metcalf is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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A technique is described for estimating the number of sheep pituitary cells remaining on Bio-Gel P2-Sephadex G-25 columns at the conclusion of perifusion experiments. After they were washed to remove growth medium, the cells were lysed by sonication in a hypotonic solution. The resultant DNA was eluted through a IO-pm filter and measured by fluorometric analysis after reaction with the Hoechst fluorochrome, H33258. DNA recovery increased in parallel with the number of cells added to a column (correlation coefficient for 52 observations on 9000-500,000 cells, 0.993; interassay coefficient of variation, 8.4%). The relevance of this technique to the general problem of counting cells in the presence of a finely divided solid phase is discussed. The loss of pituitary cells from 42 columns during a 10-h perifusion study ranged from s 10% (24 columns) to ~10-25s (10 columns), ~25-50s (6 columns), and >50% (2 columns). It is concluded (i) that pituitary cells mixed with Sephadex and Bio-Gel may be counted as DNA and (ii) that the measurement of pituitary cell loss is a necessary prerequisite for the valid interpretation of the results of peIifUSiOn experiments. 0 1985 Academic Press. II-C.

Who reads Counting cells as DNA: Estimation of ovine pituitary cells on Sephadex columns?

Author

Mary G. Metcalf

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

1985

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)