Hi,
I am working with primary cells (PBMC). Briefly, I am challenging them with different kind of factors for 48 hours.
Then, I collect the supernatant to ELISA and use the cells to FACS. I seed them in a tissue plate because I want to evaluate specific markers expressed on Monocytes, B cells, NK cells and T cells.
Does anyone know if detachment solutions can increase cleavage on membrane-bound proteins and be an issue in my ELISA measurements, as my target molecule can be expressed as membrane-bound and soluble (shed soluble). Currently, I am using accutase.
I appreciate your opinion.
Hey Rafael
Accutase does have protease activity so it may affect your cell surface protein. If it is primary cells you are working with you might want to try cold PBS containing EDTA. I've used this to lift primary macrophages before and it worked pretty well.
hope this helps
Hi,
if the earlier comment using EDTA doesn't work, you can always 'scrape' the cells from the plate using a cell scraper. You might lose some cells in the process, but you avoid using any cleavage solutions. This is what we used for long term macrophage culture where the cells do not detach using EDTA.
Hello,
I was using Accutase earlier to detach primary alveolar epithelial cells but sometimes I would lose some surface markers, so later I switched to gentle scraping and it worked better.
Using any enzymatic digestion method to dissociate cells will result in the cleavage of membrane-localized proteins. Whether this will affect your studies is up to what protein you are interested in. Most proteins, for example, have a trypsin cleavage sequence.
I agree with Matthew about using a non-enzymatic method such as EDTA or a chelator cocktail such as: https://www.thermofisher.com/order/catalog/product/13151014
Thank you all for share experience,
I will definetly avoid using ezymatic detachment solutions, I will try using cold EDTA 10mM in PBS and see how it works for my study. Then, I will post here the results.
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