I am work with some stem cells in that lab and have done my first alizarin red staining. It look like i works very well.
But i have a problem some of the other lab workers says you can put cell in a 24 well plate and let them grow for 14-21 days and just change medium every 48-72 hours. I have some problem with the monolayer growing to fast 2 good. So it makes cluds. They say cell can't live like this, and that is why i cant get it working 100% right.
Am i doing the culture of the cells before alizarin red staining right ? or do you need to do it in falsk and change medium every 48-72 hours then trysin-EDTA when confluent level is about 100 %? I cant fine any info other then people saying the change medium every 48-72 hours.
The lab has not done this work before hope someone can help.
Hello emil ,
I culture the cells in 12 well plate , i feed them 2 days / week , for example every monday/thursday or tuesday friday for 28 days and then i do alizarin red test , it works well , hope i can answer well and help you
Thank you very much. The big problem in for me is that i get this monolayer that is hard not to interupt. Have you every had that problem? I can send you a picture? I am working with adipose stem cells. But had det same problem with BMSC.
I assume you are looking for calcium deposits as an indication of cell mineralisation. If you are differentiating your stem cells toward the osteoblast lineage, then a high cell density in 24 wells (say, 100, 000 cells) from day 1 is good. I would recommend changing the medium every 48h (incl. weekends!!) for a minimum of 14 days, sometimes up to 21 days. These timings and densities of course depend on the cell type and concentrations of your differentiation components. I am not surprised that the monolayer is hard to disrupt, because of the very rich deposit of ECM being produced by the differentiating cells. There should be some clear areas where cells appear clumped (cluds?) indicating a condensation of cells.
This may also be of use:
http://www.bioon.com/z/stemcellind2015925/images/w4.pdf
Good luck
thank you very much. I am working with adipose dervied stem cells. I am seeding density of 4000 per cm2 so only around 12000 per well in 24 well palte (2,8 cm2)
I have made a pilot studie with different seeding densitys and it shows that lower is better when going for 21 days.
But thank you very much for you time.
Hi emil ,
The 24 well plate area is 1.9 cm2(~2 cm2) and it is better to take the density of cells 3000 cells/cm2 for the osteogenic differentiation . It works well with me .
And you can try to put 100 ug/ml ascorbic acid .
Good luck
Hi Emil,
As others have said, 3000 to 4000 cells/cm2 is a good number for 24 well plate to start with. And the usual protocol is to allow the cells to reach greater than 95% confluency and then start adding the osteogenic differentiation medium and replacing it every 48-72 hours.
But as you have mentioned, even I do regularly see the monolayer which gets detached while changing media and while fixing the cells. This is really tough to avoid because the osteodifferentiation causes the cells to secrete ECM and as they start depositing minerals, the monolayer is much more prone to detach (after day 14, the cells easily detaches).
You have to be really careful and very slow while changing the media or while fixing.
Good luck.
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