Hello Virginia

The final concentration of DMSO should be less than 0.1%. You have prepared a stock of 5mg/ml in DMSO. How have you made the further dilutions?

I have a feeling DMSO toxicity could be affecting your cells. Please check on this. Usually 100-300ng/ml of PMA for 48 hours should be fine for differentiation.

Best Wishes.

I agree with Malcom, you are probably seeing the effects of DMSO on the cell membranes. There is a lot of literature on the topic of THP-1 and PMA stimulation, frankly, its a little all over the place. One other suggestion is that you can try reducing the amount of PMA to 5 ng/mL as well; others will use higher concentrations, but then refresh the media after a days without PMA.

Hello, Virginia!

Please You also look at this protocol:

Article The choice of phorbol 12-myristate 13-acetate differentiatio...

2.1. Culture and differentiation of THP-1 cells THP-1 cells (ATCC) were maintained in RPMI 1640 supplemented with heat inactivated foetal calf serum (FCS; 10% v/v), Hepes (10 mM), sodium pyruvate (1 mM; all from Invitrogen) and β-mercaptoethanol (0.05 mM) (referred to hereafter as media), in a humidified incubator (37 °C/5% CO2). Cells were grown to a density of 1–8 × 105 cells/ml and used for experiments between passage numbers 5 and 10. For differentiation to a macrophage phenotype, THP-1 cells (at a concentration of 2 × 105 cells/ml) were incubated with varying concentrations of PMA (8–200 nM; Sigma Aldrich) diluted in media, in T 75 cm2 tissue culture flasks (BD) or 6 well tissue culture plates (Nalge Nunc), for 48 h. This length of exposure to PMA is commonly used in the literature (Chanput et al., 2010; Chanput et al., 2012; Park et al., 2007; Smiderle et al., 2011; Ueki et al., 2002).

I too have recently started optimising PMA differentiation for THP-1 cells we got from another lab. After a titration of PMA, I found that 10nM PMA exposure for 48h induced adherence and spindle like morphology in the thp-1 cells. However post 24 hours of rest in normal cRPMI, the cells lose their spindle morphology and some revert back to suspension. I'm completely new to this system so would appreciate feedback on how to proceed. Is a rest period in normal PMA free medium absolutely necessary before further downstream experiments?

Akansha Ganguly

Dear Akansha

In my experience, 24 hours of rest is not necessary, after 48 hours of PMA exposure, you can change the media with PMA free medium and adding your substance that you want to treat, on the same day.

Best Wishes