Hi,
I would like to perform the Clonogenic Assay in my project, and reading several protocols, they suggest to count colonies up to 50 cells. How should I recognize the 50 cells colonies?
Thank you!
Hi,
For counting colonies 1st stain them with crystal violet and then count under stereo microscope. you would be able to differentiate colonies >50 cells.
Good Luck
Hello,
Dunno what cells you are using, we do CFU assays in HSCs and follow instructions from the supplier of the kit, and consider a group of cells higher than 30 is a colony and I do know some researchers consider 60 number of cells for defining a colony.
Anyway to answer your question, for the cell type we are working with, we don't need any staining, you can easily distinguish colonies from those with less number of cells under bright field. Again, if you are not going to use the cells for anything else then staining would be OK otherwise make sure the stain will not affect future assays you are going to carry out in those colonies.
Have fun!
Hello,
I am using brightfield microscopy to analyze my colony assays. In case of doubt whether 50 or more cells form my suspected colony, I am counting the single cells within the suspected colony under higher magnification. In my case, this can be done quite easily, as hematopoietic cells under that particular condition (GCSF) do tend to form a loose cloud rather than a compact colony.
Hope this helps a bit!
Systems for automatic colony counting have been developed, but personally, I prefer direct counting under a microscope (or by naked eye based on experience) despite significant time needed for this. From my experience, through the analysis of not only surviving clonogenic colonies with ≥50 cells but also abortive colonies with
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