We are trying to differentiate THP-1 cells to macrophages and dendritic cells.
For macrophages we culture the THP-1’s with 10 ng/mL PMA for 3 days and then we rest them for 3 days more.
For dendritic cells we culture the THP-1’s with rhIL-4 20ng/ml, rhGM-CSF 100ng /ml, rhTNF 20ng/ml, Ionomycin 200ng/ml for 3 days.
We initially seed 200K/mL THP-1 cells in RPMI1640 + 10% FBS + L-glutamine before differentiation. After the differentiation we remove the cells from the flasks using trypin and a cell scraper. After counting we observer a recovery of only 10-30% (DC) and 40-50% (MQ) of the initial 2 million THP-1’s that were seeded.
Does anybody has some advice on how to improve our cell recovery? Thanks in advance!
Cells with a macrophage phenotype tend to resist to trypsin dissociation. I've used cold PBS and a cell scraper to recover differentiated macrophages before, but that reduces cell viability. Promocell have available a Macrophage Detachment Solution DXF... could you give that a try?
Best of luck!
I have observed that subculturing by scraping THP-1 or primary macrophages leads to poor viability and re-adherence from then on , I used to directly differentiate cells on the experimentation wells( which is also time saver).
Thank you all! I will look into the macrophage detachment solution. I will also try to differentiate my cells in the plates in which I will continue my experimentens. I will see if this works.
In my experience, cold PBS works well to detach macrophages, but the best for maximal recovery has been the use of Accutase (instead of trypsin), 30min at 37ºC. We have been using it for detaching macrophages and DC derived from THP1 as well as from primary monocytes and yields good numbers and viability.
hi,
There are teflon bags or biopolymer bags which can ease your problems. though i haven't used it. There are many publications where they have metioned it.
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