I;m working with THP-1 cell line. my problem is that from last ffew weaks cells are getting adhered to well plate after PMA stimulation (50 ng/ml) but didn;t get spread ir they are not showing any morphological changes like macrophage or not differentiating. Im in very problem.cells are adhereing but not differentiating and other consequence is that media colour didn;t get changed to yellow but it becomes opaque although cells are healthy. our media is RPMI-1640.
thanku.
Opaque medium? Are you sure there isn't bacterial contamination? I don't always see the morphology change, just more adherent cells. and they make cytokines too.
I culture THP1 cells for one and half year, i think it is your thp1 cell problem, final conc. at 10 ng/ ml PMA is enough to differentiate thp1 to macrophage. just chang a new thp1 cellline, don't passage more than 30 times. my experience is adhere is not very well after p40.
Similar to Anne's concern, opaque medium? May be you have bacterial or any other contamination. Next to see morphological changes, you need to incubate them for 24-48 hr post PMA stimulation. Adherance is the first property I would look for differentiation into macrophages. As Lihui suggested do not passage for more times. It also depends on how long you keep for PMA-mediated differentiation. If you want to keep for longer times, use 10ng/ml and if you are using 50-100 ng/ml of PMA keep it for 6hr to 16 hr incubations. Another issue, if you see adherance even in absence of PMA, then its either because of contamination or pyrogens in your media or glassware. Hope this helps.
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