Metaphase chromosome preparation
RPMI 1640 medium
fetal calf serum (FCS), 20%
Colcemid (e.g. Boehringer Mannheim cell biology reagents, Best.-Nr. 295892)
cell cuture flask
Phythemaglutinin, PHA-L (Seromed, M 5030)
CO2 cell culture incubator
50 ml Nunc/Falcon tubes
15 ml Nunc/Falcon tubes
KCl (0.075 M, 0.055% ?)
Fixative (methanol/acetic acid 3 : 1)
glass microscopy slides
Amounts per 5 ml blood:
40 ml RPMI 1640 Medium
10 ml FCS (20%)
5 ml peripheral blood (anticoagulation by heparin)
1.5 ml PHA
1 cell culture flask, e.g. Falcon 250 ml
prepare up to 10 flask (1 flask will yield about 50 slides)
(the acetic acid step is a washing step in particular for remowing the cytoplasm, in addition it may help for the spreading of the chromosome; the cell membranes attach to the surface of the glass slides and are disrupted by the liquid flow; the temperature difference between the cell and the glass slides may help in the disruption of the cell membranes. If the weather conditions are favorable the 70% acetic acid washing step may be omitted; in our experience the best metaphases spreads occur on dry and sunny days.
18. Air dry the chromosome slide, check for chromosome spreading and cytoplasm debris in a phase contrast lab microscope, adjust volume of fixative so that the density of nuclei/metaphases is appropriate
19. If the conditons are favorable, prepare a batch of metaphases spreads
20. Keep slide in a box at room temperature (up to about 1-2 months); metaphase spreads may be kept longer at -80°C or in 70% ethanol at 4°C.
21. Keep fixative with lymphcates at -20°C until the preparation of new slides. Add new fixative and wash cell before the preparation of new metaphase spreads.