Testing for Differentiation Blockade

Infected cells are first selected, when applicable (see Note 2), and induced to differentiate for 6 d.

The differentiation can be followed during that time period on a daily basis, but this is not an absolute requirement. Note also that no obvious difference might be apparent for the early differentiation period.

After 6 d of differentiation, estimate the percent of benzidine positive cells. At that point, control noninfected cells, or cells infected with control viruses should display at least 95% of benzidine-positive cells. Any well displaying a lower percentage of positive cells should be considered a strong indication of a differentiation blockade, especially in those cultures where the benzidine-nega-tive cells display the classical morphology of very round refringent actively growing T2ECs.

In order to definitely assess the differentiation block, simply rinse the cells and seed them back in LM1 medium. No cells whatsoever should grow out of control cultures in those conditions, and only those cells that escaped the growth inhibition associated with erythroid terminal differentiation will be able to grow. A very simple growth curve will then be sufficient to document the phenomenon.

The stringency of the differentiation blockade can be assessed from: (i) The frequency of benzidine-negative cells in the first differentiation assay; and (ii) The reinduction of blocked cells to redifferentiate (i.e., cells grown in LM1, then DM17, then LM1, then DM17 once more).

Was this article helpful?

0 0

Post a comment