staining mitotic spindle of leukocytes - a question from a total novice (Jan/08/2009 )
Hi all,
Before asking my question, I have to mention that i work in a genetics lab, doing pcrs all day long, so i know practically nothing about cell culture or immunohistochemistry.
What i am trying to do is to view the mitotic spindle human cells, as i expect to see defects. My source is a live human, living in a distant rural area, so all i can get for analysis is blood. Previously i was advised to culture leukocytes in a medium with PHA, then fixing after shortly incubating with colcemid to arrest in metaphase. But later, someone else suggested that using colcemid would destroy the spindle, and render immunostaining useless. Then I learned that other reagents such as colchicine or taxol can be used for metaphase arrest, but i couldn't find any data on how they would affect the spindle.
All that being said, what should I do? I would deeply appreciate any help or advice. Thanx in advance.
Tarik
Before asking my question, I have to mention that i work in a genetics lab, doing pcrs all day long, so i know practically nothing about cell culture or immunohistochemistry.
What i am trying to do is to view the mitotic spindle human cells, as i expect to see defects. My source is a live human, living in a distant rural area, so all i can get for analysis is blood. Previously i was advised to culture leukocytes in a medium with PHA, then fixing after shortly incubating with colcemid to arrest in metaphase. But later, someone else suggested that using colcemid would destroy the spindle, and render immunostaining useless. Then I learned that other reagents such as colchicine or taxol can be used for metaphase arrest, but i couldn't find any data on how they would affect the spindle.
All that being said, what should I do? I would deeply appreciate any help or advice. Thanx in advance.
Tarik
Using any of these drugs will greatly alter spindle morphology and therefore make your staining useless. Colcemid and colchicine both depolymerize microtubules and arrest cells in metaphase because they don't produce a spindle at all! Chromatin aligns on the metaphase plate but the spindle checkpoint is activated because there is no attachment to chromatin. Taxol stabilizes microtubules so the spindle can form but can not depolymerize after chromatin attachment. I've never cultured leukocytes so I'm not sure if they attach or are in suspension but if attached, I highly recommend mitotic shake-off as a method to isolate mitotic cells. Otherwise, the use of any drugs to synchronize may have off effects. You might be better off to just fix an asynchronous population of your cells and search for the mitotic ones. This way you have not altered the morphology or population in any way, shape or form.