I'm treating Thp1 cells with 10 ng pma and incubating for 24 hours. Is it ok or I should incubate them for more. Is it ok to give treatment immediate after PMA treatment or we should incubate in rpmi w/o PMA for sometime. I'm adding 10 ng PMA and 100 ng lps simultaneously but there is a loss of cells as comparison to treating with PMA first for 24 hours and then with lps. Kindly suggest
Different concentration of PMA mentioned for THP-1 cells differentiation with incubation time between 24-72 hrs. After differentiation of THP-1 cells into macrophages like cells, then only you can add LPS for further stimulation for inflammation model in-vitro study. Hope it will help you. You can refer few paper where i worked for same system as mentioned in below link:
Article Therapeutic Aerosol Bioengineering of siRNA for the Treatmen...
Treatment conditions would depend upon the question you are planning to ask using PMA differentiated THP-1 cells that are stimulated by LPS. For most purpose, a pre-treatment with PMA (10-100 nM) for 24-36 h (or longer) may be needed to fully differentiate cells. Medium can be changed following full differentiation to stimulate cells with LPS.
I agree with the other answers posted here. I usually stimulate THP-1 cells with PMA (100 nM) for 48 hours. I change the medium to remove the PMA and let the cells rest overnight. Then I would treat with LPS or whatever stimulus you are testing.
Dear Sandeep Kumar ,
This is the protocol that I use and it works fine:
THP-1 n 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. Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (Invivogen # TLRL-EBLPS) was added at 1 µg/mL for 4 hours to each well.
Tamara Salloum Hi Tamara, how long was the period before infection upon adding PMA-free medium?
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