21 replies to this topic

[beth]

suncherry, on Jun 30 2009, 08:48 AM, said:

Hi,
I met the similar problem  that after change the medium it detached. I have induced THP-1 into macrophage for several months. Induction methods is the same as above. Only recently the problems occured. Do you think it is because the cell itself? Because the cells have cultured and passaged for more than three months while induction methods doesn't change.THP-1 cell can passage endless? Please help.Thanks a lot.

Uh, THP-1 cells are a very stable cell line. Passage number does not matter. It has been around since the 1980's. Do you really think that you are getting passage 1 wherever you get them from? Passage number is more an issue for primary cell culture or self-immortalised cells. Just make sure you always keep stocks which are not treated with anything to bring up fresh cells when needed. I have never ever noticed anything change with passage of these cells and must have passaged them hundreds of times. I culture mine in RPMI + 10% FBS and pen/strep. Obviously, over time if you culture cells under certain conditions you may get changes, which is why results can vary from lab to lab, due to different FBS, etc.

http://en.wikipedia..../THP1_cell_line

[samasya]

beth, on Feb 5 2010, 07:43 AM, said:

suncherry, on Jun 30 2009, 08:48 AM, said:

Hi,
I met the similar problem  that after change the medium it detached. I have induced THP-1 into macrophage for several months. Induction methods is the same as above. Only recently the problems occured. Do you think it is because the cell itself? Because the cells have cultured and passaged for more than three months while induction methods doesn't change.THP-1 cell can passage endless? Please help.Thanks a lot.

Uh, THP-1 cells are a very stable cell line. Passage number does not matter. It has been around since the 1980's. Do you really think that you are getting passage 1 wherever you get them from? Passage number is more an issue for primary cell culture or self-immortalised cells. Just make sure you always keep stocks which are not treated with anything to bring up fresh cells when needed. I have never ever noticed anything change with passage of these cells and must have passaged them hundreds of times. I culture mine in RPMI + 10% FBS and pen/strep. Obviously, over time if you culture cells under certain conditions you may get changes, which is why results can vary from lab to lab, due to different FBS, etc.

http://en.wikipedia..../THP1_cell_line

I am trying to standardize thp 1 differentiation using PMA.  Can anybody tell me should I use treated or non treated plates for the treatment? and also what are the other CD markers which can be used to monitor this differentiation other than CD 14.

[Marysa]

Hi SuMi!

I am trying to differentiate THP-1 into macrophages using PMA.

I get the adderent cells after 72h of incubation.
After that time, I removed the supernantant and I also used PBS+EDTA 5mM to detach the cells. However, I just detach cells incubated with 10nM of PMA, I didn't get "free" macrophages from  (20, 40, 80 and 160nM of PMA).
I also used trypsin, and I get some detached cells, however, a lot of them continued adherent in the plate.

What do you advise??
Did I use higher concentrations of pMA, or higher incubation time?

Many thanks in advance.

Best regards

[Albatross~]

samasya, on 13 July 2010 - 05:41 AM, said:

beth, on Feb 5 2010, 07:43 AM, said:

suncherry, on Jun 30 2009, 08:48 AM, said:

Hi,
I met the similar problem  that after change the medium it detached. I have induced THP-1 into macrophage for several months. Induction methods is the same as above. Only recently the problems occured. Do you think it is because the cell itself? Because the cells have cultured and passaged for more than three months while induction methods doesn't change.THP-1 cell can passage endless? Please help.Thanks a lot.

Uh, THP-1 cells are a very stable cell line. Passage number does not matter. It has been around since the 1980's. Do you really think that you are getting passage 1 wherever you get them from? Passage number is more an issue for primary cell culture or self-immortalised cells. Just make sure you always keep stocks which are not treated with anything to bring up fresh cells when needed. I have never ever noticed anything change with passage of these cells and must have passaged them hundreds of times. I culture mine in RPMI + 10% FBS and pen/strep. Obviously, over time if you culture cells under certain conditions you may get changes, which is why results can vary from lab to lab, due to different FBS, etc.

http://en.wikipedia..../THP1_cell_line

I am trying to standardize thp 1 differentiation using PMA.  Can anybody tell me should I use treated or non treated plates for the treatment? and also what are the other CD markers which can be used to monitor this differentiation other than CD 14.

Macrophages derived from PMA treated THP-1 monocytes are very adhesive and they sticked to any surface once activated, any culture T75/T25 flask/well surface are good enough for these macrophages, so there's no need to pruchase extra collagen/matrix  treated plate for them. Apart from CD14, you can also try  common macrophage marker CD68 ...from what i did before, THP-1 cells and PMA activated  form are distinct in terms of morphology ,  protein and cytokine expressions .......no problems in distinguishing them at all

[Anula]

I treat my THP-1 with PMA. I prepared 50 ml medium RPMI with 10% inactivated serum, antibiotics solution and 50 ng/ml PMA. I would like to know If I can store this medium in refrigerator for a few days? MAybe I should do a fresh medium for every experiment….Please help

Anula

[PHVS]

I also would like to know if it's possible to store the medium for let's say, 1 month. Also, do these cells keep proliferating after differentiated?

[Anula]

Hi PHVS!

I've read a lot of papers about PMA and finally I decided to do fresh medium every time I need to. Everything works well:)

Macrophages are mature form of monocytes and they don't proliferate after differentiated.

Anula