metaphase chromosomes

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c-nem
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metaphase chromosomes

Hello;

This is my first question here. I hope I can intelligibly explain my problem.  It is about getting metaphase chromosomes from peripheral blood culture. I can't get metaphase chromosomes on my slides enough. Generaly there just a few metaphase per slide(with 5 drop) or there is no metaphase. I don't know where I make mistake. It is so complicated so it could be anything. If someone can help me about this problem I can give more details like a protocol that I follow and the images that I get from the inverse microscop. 
Getting metaphase chromosomes is the preliminary step for further analysis like FISH and chromosomal analysis. But first I should solve this problem. Help me please:) 

Biju
Biju's picture
 C-nem

 C-nem
1.Are you doing 72 hr culture or 96 hr culture?
2. What mitotic agent you are using?
3. How much of blood and media are using for culture?
4. When you add colchicine?
Std conditions need modification for each lab. So standardising each of the parameter is critical in getting good metaphases.
Biju Joseph

c-nem
c-nem's picture
Hi;

Hi;
thanks for your answer;

1. I am dooing 72 hr culture
2.I use "Phytohemaglutinin" as a mitotic agent
3.200-250 microliter blood in  5 ml "RPMI" media 

4. I add colchicine after 71 hr 20 min. later (I incubate it 37 celcius for 40 min.) 

Infact I get an image of my slides after (inverse microscope) chromosome spreading like the image "c,d,ee,h" at this web site:  http://info.med.yale.edu/genetics/ward/tavi/Slide-fig03.html.
I changed my conditions and I played with times, etc. But it still miserable.