I Hope you will find your answer in the paper attached. I followed this.

RPMI 1640 + 10% FCS+ PMA 200nM 3 day

Jitendra Kumar Shandilya Thank you for your answer.

I have already seen the paper, and the PMA concentration was slightly different, but the time went the same. However, the cells did not adhere.

The result was the same whether using 150nM or 300nM.

Can you tell what kind of cell culture plate or dish you used? Thank you.

Hello Dayoon, how are you doing today?

You are using an THP-1 cell lineage, I am afraid to say that they do not adhere in well plates, according with manufactures instructions. Also, RPMI medium is suitable for cells suspension in vitro culture and this is why most culture of leucocytes you see RPMI medium.

For THP1 differentiation, PMA could not be the best inductor of differentiation. If I am not mistaken, PMA is suitable for proliferation assays. Maybe you should try add recombinant cytokines, such IL-2 or IFN-y or LPS to induce such differentiation into macrophage.

Regarding to your last question, for macrophage makers we usually use CD80 or MHCII should be good markers for your experiment. Best regards and good luck with your research.

Tulio Teruo Yoshinaga Thank you for your kind answer.

I'll try to induce IL-2 or IFN.

Hello Dayoon

I used various protocols for the optimization of THP-1 differentiation into the macrophages (M0).

The conditions were;

• The concentrations : 5, 30, 50, 100, 150, 200 ng/mL PMA
• Treatment duration: 72 hours PMA treatment + 5 days resting, 48 h, and 24 h without resting.
• Cell density: 5 x 10^6 cells 7.5 x 10^6 cells, 10 x 10^6 cells per T25 Flask

We determined the optimal conditions for THP-1 cell differentiation into macrophages: 7.5 x 10^6 cells in T25 cm2 flask exposed to 30 ng/mL PMA for 24 hours. We used CD11b marker.

THP-1-derived macrophages (M0) differentiated towards the M1- and M2-macrophage phenotype with IFN-γ + LPS

Good luck with your experiments

Ozge Kose Thank you very much for the detailed explanation.

What was the change in the morphology of THP-1 cells differentiated into macrophages?

Dayoon Kang Cells became adherent at all conditions both in flask or well-plate. So, we didn't have any problem regarding cell adherence. We had other problems.

30 ng/mL showed the highest THP-1 cell adhesion (96%). but for example, cell adherence was found unstable at 5 ng/mL PMA concentration for 24h, and 48 h.

I hope that this information helps you

Hi! Make sure to dissolve PMA in concentrated DMSO. If you dilute DMSO to prepare PMA, you might have some trouble to differentiate your cells.

Dear Dayoon Kang ,

This is the protocol that I use and it works fine. Are your THP1 cells old i.e. in what passage are they in?

THP1 cells were cultured in RPMI-1640 medium (Sigma # R8758) supplemented with 20% FBS (Thermo # 10500064) and 1% Penicillin-Streptomycin (Lonza # 17-602E) at 37°C. THP-1 cells were stained with trypan blue and observed using the 0.1 mm Neabauer Chamber.

Prior to infection, the cells were induced to become adherent, matured macrophage-like phenotype, in 6-well plates (TPP # 92006) by the addition of 50ng/mL phorbol 12-myristate 7-acetate (PMA) from stock (1mg/mL) (Fisher # BP685-1) to the culture overnight as previously indicated. PMA-treated adherent THP1 (P-THP1) cells were washed three times and cultured in fresh medium before infection. Cell viability was determined to be >97% by the Trypan blue dye exclusion method.