Hi Abdullah,

When you say "stimulate them for next 2days for further experiments", what do you mean exactly?

Thanks!

Do you make some viability-assay with or without cholesterol treatment? We use RPMI 1640 for THP-1 cultivation and 100nM PMA for differentiation into macrophages. What for a passage have the cells?

Hi Anja,

One of the problems I find is that my cell lines comes from another, with undetermined number of passages. Although this may be important for the cells viability, in some experiments we got enough viable cells to finish the experiments while in another we did not.

This could be important?

Hi, I had problems like this before. I found out that the cells were contamined. Then I got mycoplasma-free cells and worked very well. From that point, if I start THP-1 differenciation and the cells start to die, I will new ones.

Hello Alvaro,

From my point of view and in my hands, THP-1 cells are anything but fragile.

You have to change the culture medium to RPMI 1640. We have used RPMI 1640 (PAA cat. no. E15-840) supplemented with 10% FCS Gold and 0.1 mg/ml penicillin/streptomycin/L-glutamine (PAA) for many years. We perform differentiation with 100 ng/ml PMA for 96 hours plus various periods of incubation with compounds. This protocol works fine and we detect only a few necrotic or apoptotic cells by flow cytometry (at least not more than with other cells).

Less rigid washing may also help. We split the cells usually on Monday and Friday (no washing or medium change between).

Another important parameter is cell density. If cell density during differentiation is too small, the cells behave different and are less convenient to handle. Make sure you seed the cells at around the following densities. Please also take care that the volume of culture medium is sufficient. And again: NO WASHING and NO CHANGE OF MEDIUM during differentiation.

24-well culture plate

0,38*10^6 cells per well

2 ml medium per well

6-well culture plate

1,5*10^6 cells per well

3 ml medium per well

25 cm2 culture flask

3,3*10^6 cells per flask

5 ml medium per flask

Another sometimes important point is the choice of culture flask. For some reasons macrophages grow differently in flasks from different suppliers. We have made good experiences with culture flasks and plates from TPP which are distributed by Biochrom in Germany.

Please let me know if you require any further information.

Best regards,

Stefan

Hello Alvaro,

I would agree with everything that Stefan says. We also grow the cells in RPMI but I would also predict that DMEM would work too. The important thing is to have the cells in a very healthy proliferating state before you add the PMA to differentiate them. You say that you grow them in medium containing 10% FCS for 7 days until they reach confuence. Confluence is usually a term used with adherent cells. So I suspect that the cells at the end of 7 days and "confluent" are overgrown and in poor condition before differentiation, so you might need to split the cells to keep them happily growing. Do you look at viablity before you differentiate them? We use trypan blue staining and all of the cells should be alive and look like happily proliferating cells.

We use 50 ng/ml PMA for 2 or 3 days which also seems to work very well for our needs.

Brendan

I also agree with Brendan: It is somewhat surprising that your cells grow for 7 days to reach confluence. As mentioned earlier, we usually split the cells twice a week. THP1 should be kept in suspension neither too dense nor too sparsely.

However, we tried DMEM and this does not really work fine.

Best Stefan

Supongo que hablas español, llevo años trabajando con esa línea celular y comparto tu dificultad por cultivarla o diferenciar a macrófagos, mira, yo solucioné mi problema de la siguiente manera: Primero, adiciona una concentración de 5 mM de 2-mercaptoetanol a tu medio RPMI-1640, no se que función tenga en las células pero las mantiene vivas por tiempos prolongados ademas de que no interfiere en las pruebas que necesites realizar. Otra cosa, no uses 200 nM del acetato de phorbol (PMA) usa 100 nM ya que durante la diferenciación a macrófagos suele ser tóxico y disminuye tu confluencia y viabilidad en un 60 % si excedes los 100 nM, si tienes mas dudas escribe a mi correo [email protected]

Coincido en varios puntos: el medio recomendado es el RPMI-1640 suplementado con b-mercaptoetanol. Los cambios de medio muy seguido estresan las células, además de que quitas factores de crecimiento que ellas mismas producen. Regularmente los problemas vienen cuando tus cultivos ya son viejos o tienen mucho tiempo congelados. El uso de antibióticos puede estar ocultando alguna infección. 

Con respecto a la diferenciación, son muchos los protocolos. En mi caso este artículo me puso a dudar de la metodología que estaba empleando:

http://www.ncbi.nlm.nih.gov/pubmed/24269601 

I am really new on this type of cells so do you use trypsin to split them or just pipette them up and down ?

Dear Issa,

THP-1 cells are cultured as suspension cells and can be split easily without trypsination. These cells are just centrifuged and diluted in fresh new medium at the required density.

Please let me know if you require further details.

Best regards

Stefan

Thanks a lot Stefan for your valuable answer,

Also, I have another question regarding the way we have to use to treat these cells with our compound. Do I have to differentiate them to macrophage using PMA before the treatment or I could treat them as is ?

Thx in advance

Dear Issa,

Please accept my apologies but I have not realized your new question.

Whether you treat the cells with PMA depends on your interest. If you want to study macrophages you have to differentiate them with PMA.

If youre interested in more monocyte-like cells you can treat the cells directly but please keep in mind that THP-1 suspension cells are not full monocytes.

Best regards

Stefan

Thank you so much Stefan. Another question about THP-1 derived Macrophages. Could I use them as a normal cells i.e. it could grow further and get confluent like the other attached cells or I have to use them immediately and do another differentiation from THP-1 if I want to use them again ?

THP-1 derived macropahges proliferate very poorly in my experience. If you plan to use them for experiements, you should seed THP-1 monocytes at the desired density in the final culture dish and start differentiation with PMA afterwards. From this point, cells will dramatically increase their doubling time.

Best,

Jonas

Dear Issa, Jonas is right. After PMA-induced differentiation, THP-1 cells do not proliferate anymore to a significant extent. Therefore, you have to differentiate the cells for each experiment at a defined density and you cannot continue culturing this line of cells, you have to use them immediately after differentiation (usually 72-96 h). Please let me know if you require a detailed standardized protocol. Best Stefan

How long can THP-1 derived macrophages stay as macrophages? Do they dedifferentiate back to monocyte?

Hello Stefan..

Can you please mail ([email protected]) me the detailed standardized protocol for PMA-induced differentiation of THP-1 cells. 

 We  used RPMI 1640 MEDIA for THP-1 cells.

and remain health for 3 days.

Hi Stefan,

I have just started working with these cells. Please mail me( [email protected]) the detailed protocol for THP-1 maintenance and differentiation conditions.

Dear Stefan,

I am working with THP1 cells and am doing a genome-wide screen on these cells using the CRISPR-Cas9 platform. I grow the THP1 cells in RPMI with 10% FBS and HEPES and usually seed them at a cell density of 500,000 cells per mL. I noticed that the cells usually have a baseline viability of about 85%. I tried to isolate the dead cells from the live cells using Ficoll and managed to bring the viability up to about 95%, however the viability dropped back to the 85-87% range. Do your THP1 cells have the same issue?

Hi Stefan,

I have just started working with these cells. Would you be able to please mail me ([email protected]) the detailed protocol for THP-1 maintenance and differentiation conditions?

Many thanks,

Premila

It seems THP-1 cells handling is so critical. Now, I am also so scaring because I already started seeding the cells in RPMI with 10%FBS, 0.1mg/ml AA and 0.05mM 2-Mercaptoethanol yesterday. 

I will be glad to have the standard protocol to maintain the cells. Please anybody send me the protocol ([email protected]).

With gratitude,

raihan

Thank you  Stefan! It is very important information about THP 1

Hi Stefan, 

If possible, I would be also very grateful, if you could send me the detailed protocol for THP-1 cells ([email protected]). Thank you very much!

Kind regards,

Maarja

I Recomend you RPMI 1640 medium supplemented with 10% Bovine Fetal Serum, antibiotics and 2-Mercaptoethanol for celular culture. I am using 200 nM of PMA for 72  hours and 24 hours for resting, and then start the stiumlation protocol.  I only wash three times with medium without bovine fetal serum. I hope that this information help you. Grettings

Hi Stefan,

I'm new in the use of THP-1 cells, so I'll be very gratefull if you could send me the protocol too ([email protected]).

Best regards,

Aiala

Hi Laura,

I am just starting using THP-1 cells, it would be nice and helpful if you can send me the detailed protocol too. ([email protected])

Thanks,

Sapna 

Hi everyone,

does anyone use polarized macrophages, detach them and use cytometry on them to evaluate surface markers?

If yes, specially for M1, how do you detach cells? cause M1 almost died after detachment...

Hi Clovis,

I am facing similar problem. Please let me know if you find a solution.

Thank you

I am having a similar problem. I differentiate THP-1 cells with 100nM of PMA for 48h + 24h of resting (5x10^5/well/1ml, 24-well plates). I noticed that on the first 24h of PMA stimulation, a lot of cells are floating, and when I check their viability, they are all dead and correspond to around 30-40% o the cells I plated initially. I appreciate if anyone could give me some advise.

Hi Stefan Lorkowski

I am new to the use of THP 1 cell. Could you please help me with the detailed protocol for the differentiation.

Also, a protocol with me stated 0.5 million cells, but did not state the volume of the medium or the plate to use. taolukitibi@@futa.edu.ng

Hello Stefan,

I am just starting to use THP-1 cells, it would be nice and helpful if you can send me the detailed protocol too. ([email protected]) Stefan Lorkowski

Thanks in Advance,

Wafaa

Stefan Lorkowski

Hello.

I also recently started working with THP-1.

However, the survival rate of cells after differentiation is only 50%.

Could you please send me your detailed protocol? I think it will help me a lot if you send it.

([email protected])

Thank you.

-Dayoon Kang

Stefan Lorkowski

Dear Stefan,

I hope this message reaches you. I have recently started working with THP-1 cells and all the available information in literature on these cells is quite overwhelming and confusing for me. Could you send me the detailed standardized protocol, on [email protected]? I would very much appreciate this.

Dear Stefan Lorkowski

you said you are using RPMI 1640 (PAA cat. no. E15-840) is this your standard cultere medium ?

This is just RPMI with Glutamin are you adding sodium pyruvat/bicarbonat and HEPEs to your Medium ?

Stefan Lorkowski

I hope this message reaches you. I have recently started working with THP-1 cells and all the available information in the literature on these cells is quite overwhelming and confusing for me. Could you send me the detailed standardized protocol, at [email protected]

I would very much appreciate this.

Hi everyone.

As Stefan said, THP-1 is fragile.

1. RPMI 1640 is more suitable medium than DMEM.

2. Check your cells for Mycoplasma. If the cells are infected, you see some cells have pseudopodia or are attached to the plate/flask surface and after stimulating with PMA, they die.

3. As same as the importance of cell culture plate/flask commercial brand that affects on growth and differentiation of THP-1, PMA brand plays important role. We use PMA from Santa Cruz and it is OK, but I tried Sigma and we failed to stimulate the cells. You can use from 20 to 100 ng/mL. For lower concentration you should put more time, but the the number of death/float cells are the standard range. (we use 30 ng/mL for 36 h)

4. After stimulating with PMA, change medium (a PMA-free medium) and give them at least 12 h rest, particularly if you would like to check cytokines or inflammatory pathways.

5. THP-1 cell that are kept in cryopreservation conditions as 920-950 microliter FBS and 0.8-0.5 DMSO are more resistant that those are cryopreserved with culture medium or lower volume of FBS.

I hope these are useful.

Bests,

Hamed