Counting cells as DNA: estimation of ovine pituitary cells on Sephadex columns

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 10-micron 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 less than or equal to 10% (24 columns) to greater than 10-25% (10 columns), greater than 25-50% (6 columns), and greater than 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 perifusion experiments.