Culturing blood cells is an important non-destructive approach for obtaining mitotic
material (i.e. metaphase chromosomes) that can be used for cytogenetic and
evolutionary studies. As the number of metaphase chromosomes was to low in previous
in vitro experiments with blood of the olm (Proteus anguinus), the method was further
optimized. We also included another urodele, the Iberian ribbed newt (Pleurodeles
waltl), as a model organism in the study. The primary cell culture was established from
the leukocyte fraction and from whole blood. Based on the BrdU proliferation assay, we
found that the incubation time required for lymphocyte proliferation with the mitogen
PHA-M was 4–5 days in the olm and less than 4 days in the Iberian ribbed newt, and at
a concentration of 6% (vol/vol). The appropriate concentration of the antimitotic
colcemid was 3 and 8 µg/ml, which was added to the cell culture for both 24 and 48
hours. The most promising samples for in vitro cultures were blood samples after in vivo
stimulation of cell proliferation with sequential blood sampling in a short time interval
(from 7 to 21 days). With this type of blood samples, we were able to obtain a high
number of metaphase chromosome sets in the Iberian ribbed newt (several hundred per
slide) and a satisfactory number of metaphase chromosome sets in the olm (up to 10).
However, further optimization of the procedures after in vitro culturing is required to
ensure adequate distribution and morphology of chromosomes spread for cytogenetic
analyzes.
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