There are several different methods described for CFU-F assays for MSC. E.g. in the book "Mesenchymal Stem Cell Assays and Applications" there are several partly very different methods described. Which one to choose? Where to set the cut off for a colony? 2 mm, 20 cells, 50 cells? How to seed the cells? 10, 100 or 1000 in a 6 well? Or a 10 cm petri dish? I would prefer the method Zachar et al described in Chapter 4 of the named book. What do you think?
True stem cells will form CFU with the least start up number.I recommend to start with 100 cells/10 cm.sq.This method works well for me.You can try 4 different seeding densities,maybe 100,200,300 and 500 and do a standardization yourself for the cells you are using.
All the best!
Dear Mathias,
First of all, I think the CFU-f assay is outdated for MSC research because these cells are highly migratory and therefore established colonies are likely to be the result of migration and proliferation, rather than just a measure of clonogenicity ("true stem cells") alone.
Either way - I found that a seeding density of 2 cells/cm2 isolated MSCs in P3-4 in a T-75 flask was the easiest way to do it and then to stain using Giemsa and count the number of colonies clearly visible when the flask was held up to the light. At least this method works if you are using the CFU-f to evaluate the effect various treatment conditions have on the subsequent colony count.
Best regards,
Ina Laura
@ charan thej
did you mean 100 cells / cm2 ??
because i think 100 cells / 10 cm2 is very low density no ?
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