Hello

i agree with Gintautas Saulis  100% , look at link s here

https://www.nature.com/nprot/journal/v1/n5/full/nprot.2006.339.html

https://www.researchgate.net/publication/6416427_Clonogenic_assay_of_cells

will help you , Good luck

Article Clonogenic assay of cells

Ideally, for a true clonogenic assay, start with one or two cells dissociated from neurospheres or embryoid bodies in each well of a 48-well dish. A larger diameter well is not more beneficial; the stem cells, once dividing, like close proximity. Then perform daily cell counts and sphere counts. Change the media very carefully and do not passage during the clonogenic assay.

Being in "close proximity" would defeat the purpose of clonogenicity. Try doing a limited dilution technique to identify the optimal number of cells for whatever plate size you are attempting to use. We used this method on 100mm culture dishes and we were successful. Hope this helps!

One can produce > 5x conditioned medium and plate one cell in a tissue culture vessel. It doesn’t matter whether vessel is one well in a 96-well plate or a T-175 flask. Single cells respond to paracrine exosomes secreted by adjacent cells. One can “fool” a single cell into “believing” that it is surrounded by neighboring cells by using 5x or greater conditioned medium. We used this technique to clone from a single cell: mesodermal stem cells [Anat Rec 263:350-360, 2001], pluripotent stem cells [Anat Rec 277A:178-203, 2004], and totipotent stem cells [Minerva Biotech 17:55-63, 2005]. This is the reasoning behind the "close proximity" eluded to by RP Singh.

I agree with Dr Young on this. In the past I had single cell cloned MSCs in 96 well plates in conditioned medium. However, I used mouth pipetting and a microscope. Approximately 80% of wells had individual colonies. Duplicates discarded. Some wells gave no colonies. You have to check the wells at several times over to ensure only single cells are seeded.

If you are lucky enough to a a FACS sorter that can seed individual wells, this is somewhat faster (after all the set-up). My colleague recently did this with about 90% efficiency.

Whatever the choice, conditioned medium is certainly a good way to go. I don't know what Rock inhibitor might do to MSCs because I never used it, but have a read to see if it is compatible for you. I use it for hESCs.

Substrate choice might also help. Of course you can use plastic, but that will only capture a subset of cells - ie, those that can attach to plastic. Not all cell types do. You can use it as part of a selection process, or change this by using a general substrate like Matrigel.

Finally, from my experience (unsorted) MSCs are highly heterogenous populations. Several cell types appear. These have different morphologies, growth rates and differentiation potentials. Fibroblastic morphology grew the fastest, for more population doublings, and had better classical MSC tripotent differentiation. Other cell types had cobblestone morphologies, and some increased in cell number, but were not forming monolayers...they just divided and sat alone, but near each other.

You'll have to do this for yourself to find out what you have.

I did much of the work manually, with few tools. These days you have many more options.

This might sound simplistic, but I used a different coloured pen to label each type of morphology. You can count hundreds of colonies. I personally did nearly one thousand this way. If you want to passage them, then it gets slower. Even still, you can passage 50 - 100 clones quite easily by hand. It will take you a whole day, but you'll have the cells growing and can do more with them.

Again, conditioned medium is important. Ideally from rapidly growing cultures (25-85% confluence. In general, don't let your cells ever get confluent without reason.

Good luck.