Method for metaphase spreads: can you help?

Engelbert Buxbaum EBUXBAUM at man.ac.uk
Wed Oct 18 06:33:50 EST 1995


In article <45gfgl$s3p at clus1.ulcc.ac.uk>, g.coulton at cxwms.ac.uk 
says...
>
>Metaphase Spreads: can you help?
>Can you help me by sending me a good robust method for 
preparation of 
>metaphase spreads from peripheral blood samples?  If you can 
many thanks 
>in advance.
>
>Gary Coulton
>
>
The most important thing is to have absolutely clean slides 
(chromium sulfuric acid and several changes of aq. bid., than 
handle them with gloves only. Store in icecold water). The cells 
(after treatment with colchicin for 24 h) are washed and 
resuspended in hypotonic buffer, so that they swell (carefull, 
at this stage they are very sensitive). Then spin down and 
resuspend in icecold Carnoy's solution. Take the suspension 
(still in Carnoy's) up in a pasteur pipette and  let single 
drops fall onto the slides from a hight of about 30-50 cm. The 
cells will rupture and the chromosomes spread onto the slide, 
provided it is absolutely grease-free. Dry with a blow dryer and 
use Giemsa to stain. I have done this successfully with both 
blood samples (stimulated with phytohaemagglutinin) and with 
cultured cells. There are only two pitfalls: if the slides are 
not clean, the chromosomes will lump together, and when the 
swollen cells are not handled carefully, they will break before 
you drop them onto the slides. 




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