Standard procedure to differentiate THP-1 cell is treatment with PMA and selection of adherent cells. Is there any specific markers that can be used to confirm the differentiation process, or that could be used as a tool to estimate the stage of differentiation?
Hi!
Been there, I assume you're using 100 nM, we have used CD206 in the past with 50-60% differentiation by flow cytometry. CD11b is what we've switched to right now and we have seen good results with similar ratios so far, but we haven't finished fully testing. Let me know if I can help with anything else.
CD36 is a good marker for THP-1 differentiation and is upregulated upon differentiation. What concentration of PMA do you use and for how long. Be careful that your PMA treatment does not interfere with your test conditions. PMA treatment as low as 5ng/ml for 48 hours has been shown to suitably differentiate THP-1 cells (http://link.springer.com/article/10.1007/s00011-007-6115-5#)
You can allow the treatment with PMA even for 72hrs and compare the results at different times of treatment with PMA., As JOSE RUFFINO suggested CD36 is a good marker for THP-1 differentiation. lalitha kabilan
Also keep in mind that not all macrophages are the same. The type of macrophage you get will depend on what differentiation protocol you use. There are different protocols that are designed to give you M1-type or M2-type macrophages. Often these are used after differentiation with PMA.
Hi Nicolas,
I would suggest CD11b (postive at 98% within 24hr PMA treatment 5-10 ng/ml); Cells will also become CD54 (ICAM1) positive. Note that PMA treatment has been reported to cleave CD14, you can get CD14 expression using vitamin D3 treatment (4-5 days) but the cells are reported to be monocyte-like rather that macrophage-like.
Good Luck, Claudie
Thanks all for the answer.
Apparently, best markers for differentiation would be CD11b and CD36. Is it possible to measure them by qPCR, or is FACS the only way?
Hi Nicolas, you can measure using qPCR but this does not always relate to what is occurring at a protein level so if you want to ensure that the cells are fully differentiated, it is best to look at protein levels via FACS, fluorescent microscopy or Western Blotting. There are other methods that you can adopt, like looking at the morphology of the cells under a microscope (which is quick and easy and can be combined with fluorescent microscopy) or looking at functional properties of the cells which is a bit more complex e.g. phagocytic ability but this depends on how important this is to your investigations.
CD11b and CD36 are the usual markers for this.
For different cytokine profiles upon stimulation of the THP-1 derived monocytes and macrophages:
Chanput, W.; Mes, J.J.; Vreeburg, R.A.M.; Savelkoul, H.F.J.; Wichers, H.J. (2010)
Transcription profiles of LPS-stimulated THP-1 monocytes and macrophages: a tool to study inflammation modulating effects of food-derived compounds
Food & Function 1 (3). - p. 254 - 261.
Hi Nicolas, have you considered using a functional flow assay to evaluate the differential status of your THP-1? We have used a combination of surface antigen expression(eg CD36) and phagocytosis of opsonised fluorescent latex beads to assess macrophage differentiation in THP-1
Hello Nicolas,
I refer me and my collegues (Tompa et al 2011):
In non-differentiated THP-1 promonocytes, during respiratory burst the
level of superoxide radical production so low that it
has been ignored in most studies. Instead, the respiratory burst measurements
have been performed using differentiated cells. The faint superoxide
production in non determined THP-1 cells is due to the low-level expression
of functional NADPH-oxidase complexes and phagocytosis
receptors like cd11b. Upon differentiation, the THP-1 cells express increased
NADPH-oxidase activity, cytochrome b content and cell membrane
CD11b receptors and acquire manifold capacity to produce
superoxide radical during respiratory burst (24–27). Indeed, the appearance of
RB has been used as an indicator ofmaturation of THP-1 cells and
leukemic cell lines PLB 985 and HL 60 (28).
Tompa G., Laine A., Pihlanto A., Korhonen H.J., Rogelj I. and Marnila P. (2011). Chemiluminescence of non-differentiated THP-1 promonocytes: developing an assay for screening anti-inflammatory milk proteins and peptides. Luminescence 2011L; 26 (4): 251-258. DOI: 10.1002/bio.1220
24. Dugas N, Mossalayi MD, Calenda A, Léotard A, Bécherel P, Mentz F,
Ouaaz F, ArockM, Debré P, Dornand J, Dugas B. Role of nitric oxide in
the anti-tumoral effectof retinoic acid and 1,25-dihydroxyvitaminD3
on human promonocytic leukemic cells. Blood 1996;88(9):3528–
34.
25. Schwende H, Fitzke E, Ambs P, Dieter P. Differences in the state of
differentiation of THP-1 cells induced by phorbol ester and 1,25-
dihydroxyvitamin D3. J Leukoc Biol 1996;59(4):555–61.
26. Gross A, Dugas N, Spiesser S, Vouldoukis I, Damais C, Kolb JP, Dugas
B, Dornand J. Nitric oxide production in human macrophagic cells
phagocytizing opsonized zymosan: direct characterization by measurement
of the luminol dependent chemiluminescence. Free Radic
Res 1998;28(2):179–91.
27. Almeida AC, Rehder J, Severino SD, Martins-Filho J, Newburger PE,
Condino-Neto A. The effect of IFN-gamma and TNF-alpha on the
NADPH oxidase system of human colostrum macrophages, blood
monocytes, and THP-1 cells. J Interferon Cytokine Res 2005;
25(9):540–6.
28. Ashkenazi A, Marks RS. Luminol-dependent chemiluminescence of
human phagocyte cell lines: comparison between DMSO differentiated
PLB 985 and HL 60 cells. Luminescence 2009;24(3):171–7.
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