The human leukocyte test system. V. DNA synthesis and mitoses in PHA-stimulated 3-day cultures

In human leukocyte cultures st up with TC medium 199,DNA synthesis and mitotic indices were analysed by means of 3H-thymidine autoradiography and cell counting. DNA synthesis starts at around 28 hrs. The frequencies of labelled cells rise slowly and reach a maximum of around 24%. The first mitoses appear at around 38 hrs but up to 49 hrs only very few mitoses can be seen. After that time the mitotic indices rise and reach values of up to 11% cultivation in the presence of BudR for 72 hrs and staining with Hoechst 33258 stain revealed that first, second and third mitoses occur together in the cultures at this time. Irradiation of whole blood and cultivation for 72 hrs leads to mitoses containing dicentric and ring chromosomes with and without fragments, to interphases with micronuclei, to premature chromosome condensations (PCC) and to polyploid mitoses indicating that at this time first and further mitoses are present.