I want to staining the live cell colonies is soft agar assay. My lab we have INT(Iodonitrotetrazolium chloride) it's in solid state, the catalog is I8377 sigma. I want to know how to prepare it for staining cells in 6-well plate. what's concentration should be used and how much amount to add. and how long the staining takes.
And my second question is 'is there any other method to use for staining cell colonies in soft agar'. I saw some people using crystal violet, but the preparation and staining protocol is very diversity. So I want to ask who has experience in this assay and maybe show me some of your figures.
Thank you very much in advance!
Dear Jun,
you can find some information concerning solubility in the Product Information Sheet (see: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Product_Information_Sheet/i8377pis.pdf)
Solubility in water is 4mg/ml, higher concentrations are possible using ethanol or dimethylsulfoxide.
I have always used crystal violet for staining colonies growing in soft agar, the procedure is really quite simple. There are several reports describing the method (see: http://web.mnstate.edu/provost/Soft%20Agar%20Assay%20Protocol%202012.pdf ; http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=104)
Good luck with your experiments,
Christian
0.1% of crystal violet for 20-30 min staining can do this job very well.
There is always the alternative of an automated colony counter where stains are generally not required to see and count colonies. Here is a webinar on some of the merits of automated colony counting.
https://www.scintica.com/webinar-automated-colony-assay-counting-advancing-the-frontiers-of-cancer-research/
If you want to stain alive cells could be better to stain them with MTT, viable cells with active metabolism convert MTT into formazan. Dead cells, on the other hand, lose this ability and therefore show no signal. Crystal Violet does not distinguish dead from alive cells, CV only distinguish between attached and detached cells. Cells into soft-agar are confined in it.
Please, try to adapt this simple protocol:
1. Remove carefully the media over soft-agar.
2. Wash with PBS and remove again.
3. Add 200 µl (p24 wells) of MTT 1 mg/ml in PBS.
4. Incubate overnight at 37°C
5. Remove carefully the MTT staining solution, wash with PBS, remove again.
6. Colonies will appear dark brown color, then yo can take photografs of wells.
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