Hi! I am doing flow cytomety experiments on monocyte derived macrophages. I tried to use accutase which should not affect macrophages surface markers. However, I can detach no more than 50% of seeded macrophages, even after increasing the time of detachment to 20 minutes and washing cells twice with PBS w/o calcium and magnesium before using accutase. What else I can do to improve macrophage detachment?
Hello Patryk Firmanty
Yes, you should use accutase as it is a popular alternative to trypsin, being much gentler on surface proteins while at the same time not compromising detachment efficiency.
Before dissociation, wash the cells with PBS (w/o calcium and magnesium) + 0.1 mM EDTA to deplete the cells of bivalent cations. Since no more than 50% of seeded macrophages get detached, you may want to try other alternatives to enzymatic dissociation for cell detachment. You may use 5mM EDTA (for 15-20 mins). EDTA complexes bivalent cations that are needed for effective cell adhesion. EDTA may also be used in combination with cold shock at 4°C to enhance the dissociation effect.
Also, there is an additional non-enzymatic method that you may want to use namely, the use of commercial detachment media that are designed specifically for the dissociation of macrophages from cell culture plates. But many of these come with relatively high costs and little reference data is available regarding their composition or efficiency.
Best.
We have the same issue that not all macrophages detach, so we scrape the remaining cells off while they are still in the accutase buffer. The viability remains high, above 90%.
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