Optimising seeding of normal and FOP cells for a later assay – prettier than expected

Further down the line I want to be able to test the activation of the BMP signalling pathway downstream of the ALK2 receptor. For this I plan to use the alkaline phosphatase gene as a readout – it’s a well known gene activated downstream of BMP signalling so a lot of easy to use assays already exist for it.

Unfortunately when I tested this assay with the standard C2C12 cell line it didn’t work particularly well so I’ve decided to try again with a new set of cells. This time I’m choosing connective tissue cells (fibroblasts) from FOP patients as a reporter cell line because they respond more strongly to the signals that activate the BMP pathway (called BMP ligands).

I’ve never worked with these cells before so, again, I needed to work out how many I’ll need to get a good result from the assay. These cells however grow as a very thin sheet which is so see-through that I initially couldn’t tell if the wells were full. To solve this I used stains called Calcein AM and Hoechst. Calcein AM can stain any living cell because they metabolise it from a see-through molecule into a fluorescent green product, whereas Hoechst can stick to DNA and turn all the cell nuclei blue, and so suddenly, all of my cells glow:

Fibroblast cells stained with Calcein AM (green) and Hoechst (blue)

Fibroblast cells stained with Calcein AM (green) and Hoechst (blue)

Aren’t they pretty?

Once they’re this easy to see, it’s even easier to see how many cells are needed to fill a well, but not get overcrowded. You can read more about how I did that in my Zenodo post.

The only question I have left is why some cells didn’t stain with Calcein AM, even though their nucleus looks fine? Anyone know the answer?

One Reply to “Optimising seeding of normal and FOP cells for a later assay – prettier than expected”

  1. Hi Elizabeth! I am working on developing new inhibitors targeting Alk2. I however do need help choosing the correct cell lines. Do you think we chat sometime?

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