Help with karyotyping - (Mar/04/2009 )
I have been working on establishing a primary cell line over the past 2 months using an experimental procedure. While the cells look like they are the correct cell type, I want to double check to make sure contamination has not occurred. I tried growing my cells on a coverslip, arresting them with colcemid, incubating them in a hypotonic solution, then doing a giemsa stain. While the cells grow on the coverslip, and I am able to visualize them, I am having trouble counting the actual number of chromosomes, as they are so condensed. Does anyone have any tips that might help make this process more sucessful. I welcome suggestions, however I would also not be opposed to trying an entirly different proticol as long as it seems plausable.
If you are just trying to identify the cells have you tried to immunostain them for cell specific markers?
If your not familiar with this basically it uses primary and secondary antibodies which are specific for proteins that only your cell typew will produce (can be enzymes or other proteins specifc to cell type)
You can normally find information on this on other papers published on the same cell type or even a whole lot of markers if you can find a paper where they developed a cell line from the exact same cell type
By exposing the cells to the primary antibodies for the particular protein markers (can buy these from santa cruz although other memebers may know of better companies)
And then exposing them to a secondary antibody with a stain such as FITC or texas red you can visualise the cells using a fluorescent camera to see if they have that particular marker, thus if they are the cell you think they are.
If the cells do not fluoresce then they dont have that protein and are probably not the cell you think
Hope this helps.
The whole procedure is very easy and takes about a day to perform.
The antibodies usually cost around 250 dollars each (could always ask for trial vials and get a few for free) but other reagents are very cheap apart from the fluorescent camera which most labs would have