I used flow cytometry for determination of ploidy level but I didn’t analyse standard sample.
Standard or reference whatever we called are important to determine ploidy level. But there are two type of reference : internal and external. Internal is the better, but if you choose external, maybe you can still compare one or two samples from your run with the reference and then maybe you can use the calculated value of them to find the others' value of the others, or you may renew your run with reference otherwise you never know the ploidy of your samples. Which type of machinery you are using: tube-based or high throughput?
It will depwend on your sample. For instance, for pure tumor cells (i.e. ALL or CLL with >95% tumor cells), a reference healthy sample is reccessary. For infiltratred tissues (hoimogenized tumors, Bonbe Marrow o MM,...) the mixture between normal and tumor cells will allow to distinguish between the two types of ploidy easily.
Thank you for your guide Dr. Serdar Dirihan
I want to use external standard (reference) but I don’t know how compare them.
G1 in my sample = 69.72
I think you can renew your samples with the reference in the same day even in the same hour because fcm machines can generate different value for different times , external standard might be useful to check chromosome duplications but for aneuploidy (like one-two chromosome differences) internal standard is better. Your G1 peak value is belong the day when you run your samples and also to avoid artifact standard and samples should be run at the same run, otherwise you couldnt compare. Beside flowingsoftware.com, the following thesis is on internet well-explained for fcm in plant sciences http://www.ibot.cas.cz/fcm/suda/presentation/disertation.pdf, have a good work!
You need to search literatures to understand the diploid (2n) level of any plant species before you can subsequently determine and identify varying ploidy levels- tetraploid, triploid, hexaploid etc. For example in Stevia rebaudiana Bertoni, a plant species, the 2n=22, but other ploidy levels like 3n=33, 4n=44 heve been found in nature. Thanks.
Yes, you can estimate the ploidy level for particular tissue or organ in one time because the peaks generated by FCM module are based on nuclear content of higher or lower ploidy cells compared to normal ones. But for publishing purpose and to justify the validity, the results will be of no use without reference sample.
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