PMA induced U 937 differentiation to Macrophages - (Oct/05/2005 )
Hi,
I Would like to ask if anybody knows in what concentration of PMA and in how many cells/mL U937 monocytic cell line is differentiated to macrophages. Yet, how many days are required for the differentiation to take place.
During differentiation should I expect any morphological change under microscopic observation or these cells are attached mantaining their round shape?
Thanks!
I have used vitamin D3 and TGFB to differentiate these cells. You will notice that they adhere upon differentiation.
Thanks a lot!
Hi Babies,
we are using PMA for THP-1 differentiation after treatment we geting cell differentiated. Here we were using 20nM PMA /ml media and incubates for 14 hrs then after 14 hrs replacing media by fresh RPMI media for further 58 hrs totat time requres for fully differentiated cell is 72 hrs.its not required how much cell you are using.
You can take this much amount for U937 cell line perhapes its work on this also, you can check cells viabilty after 72hrs by trypan blue assay if you geting more blue cells then you can decrease the conc of PMA.
For futher any quiary you can ask without any hesitation we are using PMA from last 2 years for cell differentiation.
ok
Best of luck
awadh
I have used vitamin D3 and TGFB to differentiate these cells. You will notice that they adhere upon differentiation.
Thanks a lot!
I seed 7 * 10E5/ml in a 24 wells plate (1ml/well). Add 10nM PMA for 24 hours, wash cells and replace for regular medium (RPMI, 10% FCS). Then let them differentiate for another 48 hours. The cells will adhere and grow in size. You will need to remember that you do activate these cells with PMA!!! They will start producing cytokines such as IL-8 in high amounts. However it is still possible to increase cytokine production with a high dose of LPS (1 microg/ml). For comparison, I use 1 ng/ml to activate primary human macrophages.
Thank you very much, your information are realy helpful!
we are using PMA for THP-1 differentiation after treatment we geting cell differentiated. Here we were using 20nM PMA /ml media and incubates for 14 hrs then after 14 hrs replacing media by fresh RPMI media for further 58 hrs totat time requres for fully differentiated cell is 72 hrs.its not required how much cell you are using.
You can take this much amount for U937 cell line perhapes its work on this also, you can check cells viabilty after 72hrs by trypan blue assay if you geting more blue cells then you can decrease the conc of PMA.
For futher any quiary you can ask without any hesitation we are using PMA from last 2 years for cell differentiation.
ok
Best of luck
awadh
Hi awadh,
thank you very much for your reply!
I wonder if you could answer to some further quetions.
First of all is the 20 nM the concentration of PMA you use or is it 20 ng/mL of PMA (I ask because the 20 nM/mL is confusing due to /mL).
I would also like to know if the differentiated cells continue to grow in number after replacing the medium with PMA with fresh medium without PMA.
Finally, do you use any specific assay to confirm the differentiation of U937 to macrophages? (immunohistochemisty? or something else?)
Thank you in advance!
I have used vitamin D3 and TGFB to differentiate these cells. You will notice that they adhere upon differentiation.
Thanks a lot!
I seed 7 * 10E5/ml in a 24 wells plate (1ml/well). Add 10nM PMA for 24 hours, wash cells and replace for regular medium (RPMI, 10% FCS). Then let them differentiate for another 48 hours. The cells will adhere and grow in size. You will need to remember that you do activate these cells with PMA!!! They will start producing cytokines such as IL-8 in high amounts. However it is still possible to increase cytokine production with a high dose of LPS (1 microg/ml). For comparison, I use 1 ng/ml to activate primary human macrophages.
Thank you very much, your information are realy helpful!
I wonder if you could tell me if the differentiated cells continue to grow in number after replacing the medium with PMA with fresh medium without PMA.
Do you use any specific assay to confirm the differentiation of U937 to macrophages?
Thank you in advance!
The cells should stop dividing. I have never measured surface markers to check maturation, I just looked at morphology. You could perform FACS analysis for differentiation markers if you want to be sure.
Dear awadh,
I am just using PMA for induction of J774a.1. I always see inducing the cell with 0.3uM for 72 hours in the protocols. Do you mean that the induction just require a few hours, and let the cell grow in fresh DMEM with FCS is OK ? I have problems in inducing the cell. I have incubated the cell with the DMEM/FCS and PMA together for 72 hours in total, first , I have incubated the cell for 48 hours, then scarped the cell from the flask and seeded them into 24 wells plate. I always find that many cell die after scraping from the flask and not all the cells are differentiated into macrophage.
Kenc
we are using PMA for THP-1 differentiation after treatment we geting cell differentiated. Here we were using 20nM PMA /ml media and incubates for 14 hrs then after 14 hrs replacing media by fresh RPMI media for further 58 hrs totat time requres for fully differentiated cell is 72 hrs.its not required how much cell you are using.
You can take this much amount for U937 cell line perhapes its work on this also, you can check cells viabilty after 72hrs by trypan blue assay if you geting more blue cells then you can decrease the conc of PMA.
For futher any quiary you can ask without any hesitation we are using PMA from last 2 years for cell differentiation.
ok
Best of luck
awadh
I have used vitamin D3 and TGFB to differentiate these cells. You will notice that they adhere upon differentiation.
Thanks a lot!