karyotyping!!!!!!

[michel_mr]

we know that karyotype is used for example to find trisomy right? so how we can be sure about it if the sample we take from the fetus or adult may contain more than one cell,so it contains more than 2 chromosomes from one pair,can any one help me ?

[CharonY]

The chromosomes between cells are not mixed during the process.

Essentially the cells are fixed, spread (so that they don't overlap) and dried. The chromosomes are then effectively immobilized and the nuclei of the different cells are apart from each other.

[michel_mr]

thx but i am sorry i dont get it,when we prepare the process we take wbc in culture and we rupture their membrane by hypotonic solution so we could have access on chromosomes then we take few drops on slide.......... so i can get it till now how we could we determine the trisomy if we are using many cells in suspension

[CharonY]

Could you post your complete (and precise) protocol? It may be different from those that I know.

Also, are you sure that you already lyse the cell in the hyptonic solution or do you just let them swell?

[michel_mr]

1- take sample of blood then after culturing WBC we stop division at metaphase by colchicine. then we add hypotonic solution to rupture cell membranes,after we take few drops on slide with dyes and photo ......

it is from a book and i cant get it at all,i wish some lab doc or technician can help me. hope u can

[CharonY]

Do you drop the sample from a slight height onto the slide?

Most protocols for blood use hypotonic buffer to lyse the red blood cells. White blood cells are fixed and spread via dropping them onto a slide. If you lyse them beforehand I'd assume that the nucleus should maintain enough coherence to have the chromosomes lie together. In any case the idea is that by dilution and spreading the given nuclei become separated.

[michel_mr]

cells during metaphase of mitosi dont have nuclear membraneand the are in the cytoplasm . thx but i am still confusing:eek:

[CharonY]

Right you are, my mistake. However, I can only reiterate, usually the hypotonic solution does not lyse the cells but merely swells them. You then add fixatives (e.g. acetic acid methanol solution) to maintain the cells in that state and makes the membrane instable. Then you just spread the cells on the slides. As I said, a precise description of your protocol would be helpful for any discussions.

Alternatively check this article:

Cytometry 2001, Vol 43, p101-109