Iam using PMA at 25 ng/ml for 24h followed by washing and incubation for another 24h in fresh medium (RPMI+10%FBS) to differentiate THP-1 monocytes. Within this time duration, I definitely observe that all the cells are adhered which is expected once THP-1 cells differentiate into macrophages. What Iam wondering is whether they undergo a change in morphology as well, that is do they become larger in size or elongated etc.
Hello Anjali,
I have to disagree a bit with Ashley. The effect of PMA on THP-1 monocytes differentiation will depend on PMA concentration and exposure time. After thorough bibliographic search, I've seen researchers use PMA from 10 to 500 ng/mL, and for 24 to 72 or more hours. Both described and in my own experience, PMA as low as 10 ng/mL will, as you have seen, turn a good percentage of the monocytes adherent, nevertheless full differentiation into M0 macrophages will take at least 40 hrs. At this time, not only most of your cells will be attached, but they will also flatten, grow, and increase the citoplasm:nucleus area ratio. You can also check this differentiation by analyzing CD14 expression. I suggest reviewing a paper published by Park et al. 2007 "Optimized THP-1 differentiation is required for the detection of responses to weak stimuli".
I hope you find this useful, best regards,
Martín
Hi Anjali,
I advice to add Vitamin D3 in addition to PMA in order to induce the cells differentiation
Hi Anjali
as mentioned by others, PMA conc,and time of incubation but would like to add based on personal experience the quality of cell culture plates. If we use normal cell culture plates we do see attachment of THHP-1 cells but they tend to remain more towards circular and little flat morphology,however, we we used cell bind plates we could clearly see the extended morphology typical of macrophages.
My input may not be as helpful, but there are a few tips I can provide!
From my experience, Macrophages can be induced with exposure of 18-24 hours of 100 ng/mL PMA. Following this, you can change the media, and scrape the cells to collect enough macrophages for your purposes. If you need to transfer cells for whatever reason, please use un-treated Petri Dishes!!! These cells will hold on for dear-life, and I have lost cells because of this. They would not come off when I tried several different extraction methods. So, the key with working with Macrophages would be to differentiate them on untreated surfaces, so that the cells can be extracted for future tests. And it seemed to have been okay to work with such a high PMA concentration for my lab's purposes, as we only let them stay in contact with PMA for less than 24 hours.
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