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Help with THP-1 cells


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#1 BryanC

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Posted 06 April 2010 - 10:56 AM

I have a few questions about these cells.

Is this cell line truly monocytic or or are they a some precursor to monocytes?

ATCC lists them as "monocytes" but Ive also seen them refered to as pro-monocytic. Is PMA only enough to induce to a specific polarization of macrophage? When trying to induce these cells to various macrophage activation states, is it necessary to first induce differentiation with PMA and then introduce cytokines to drive polarization in a certain direction? Or is induction with say IL4/IL13 or with IL10 enough to elicit a phenotype change? I have come across papers that seem to have contradicting methods.

Also in your experiences, have you continued to use 2-ME in the induction medias or only in the original growth media? Would 2-ME affect differentiation?

TIA for any help.

B

#2 jakatta70

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Posted 06 April 2010 - 11:15 AM

Hi,

I used to work with THP-1 monocytes in my first postdoc and I was interested in differentiating to classically and alternatively activated macrophages. I tried both PMA for 3 days and then specific cytokines thereafter as well as cytokines alone to induce distinct phenotypes. I found that cytokines alone was not enough to differentiate from monocytes to a specific macrophage phenotype. So Id advise PMA at 20nM for 3 days, remove media and then add your cytokines.

Im unsure about the mercaptoethanol question. Its function in media is to break down toxic metabolites from the cells. Id presume then that the small concentration of BME in your media wouldnt affect macrophage phenotype but I cant say for sure. You could contact Dr Judith Allen at the University of Edinburgh, Dr Sheila Donnelly at Brisbane or Dr Derek McKay at University of Calgary. They would easily be able to answer any questions on macrophages for you.

#3 BryanC

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Posted 06 April 2010 - 12:17 PM

Thanks for your reply. So when you treated with PMA did the cells attach and/or change morphologically? Im assuming they definitely did after the cytokine treatments. How lond did you treat with cytokines? Did you have any post induction growth period with RPMI 1640?

I was just thinking outloud with the 2-ME. I didn't know if it could effect the disulfide bonds on the cytokines I am using for induction, although it is only at .05M so probably not.

Also, thanks for the references!

B

Hi,

I used to work with THP-1 monocytes in my first postdoc and I was interested in differentiating to classically and alternatively activated macrophages. I tried both PMA for 3 days and then specific cytokines thereafter as well as cytokines alone to induce distinct phenotypes. I found that cytokines alone was not enough to differentiate from monocytes to a specific macrophage phenotype. So Id advise PMA at 20nM for 3 days, remove media and then add your cytokines.

Im unsure about the mercaptoethanol question. Its function in media is to break down toxic metabolites from the cells. Id presume then that the small concentration of BME in your media wouldnt affect macrophage phenotype but I cant say for sure. You could contact Dr Judith Allen at the University of Edinburgh, Dr Sheila Donnelly at Brisbane or Dr Derek McKay at University of Calgary. They would easily be able to answer any questions on macrophages for you.






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