Researchers are facing difficulties during in vitro expansion of undifferentiated hematopoietic stem cells (HSC). PromoCell Hematopoietic Progenitor Cell (HPC) Expansion Medium DXF provides a highly optimized, chemically defined, and xeno-free culture system for human HSC. 
We offer a step-by-step protocol for the in vitro maturation of human monocytes into immature monocyte-derived dendritic cells (moDCs), as well as into fully mature moDCs, using PromoCell-media. 
Here we describe how the presence of PromoCell differentiation media encourages in vitro differentiation of mononcytes to macrophages. These then show either an M1-like or an M2-like polarization. 
Primary human macrophages are difficult to isolate and do not proliferate in culture. PromoCell offers a complete system for easy and cost-efficient generation of pure monocyte-derived macrophages. 
Monocyte Attachment Medium
Defined and xeno-free medium for efficient adherence selection of monocytes from freshly isolated human mononuclear cells.
Monocyte Attachment Medium
C-28051
250 ml
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The Monocyte Attachment Medium allows for the efficient adherence selection of monocytes from freshly isolated human mononuclear cells while maintaining optimal cell health. Thus, the time-consuming and costly immunomagnetic purification of monocytes is dispensable prior to experiments requiring adherent monocytes as a starting material.
With properly performed washing steps (see Instruction Manual) large numbers of 80-90% pure monocytes can be obtained with the PromoCell Monocyte Attachment Medium. Typical applications are generation of dendritic cells / macrophages as well as phagocytosis assays.
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Technical Library (10)
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Why is it not necessary to use Monocyte Attachment Medium (C-28051) when using DC Generation Medium DXF (Ready-to-use, C-28052)?
The DC Generation Medium DXF (C-28052) is recommended for freshly isolated monocytes or mononuclear cells. When using this medium, cells will attach efficiently, so that an additional medium like the Monocyte Attachment Medium (C-28051) is not necessary.
In contrast, when using the DC Generation Medium (C-28050) for freshly isolated monocytes or mononuclear cells, the Monocyte Attachment Medium (C-28051) is needed for an efficient attachment.Related Links and Documents
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We use Macrophage Generation Media from PromoCell to get differentiated macrophages from peripheral blood monocytes. The monocytes are isolated by culturing PBMCs in Monocyte Attachment Media (also from PromoCell) for 1.5 h. Would it be ok if the attachment step is done overnight, since we receive the blood later in the day which makes it difficult to process on the same day?
It is not recommended to leave the blood cells in the Monocyte Attachment Medium for longer than 1.5-2 hrs. The medium was developed for (short-term) attachment of the monocytes and does not provide nutrients for a longer time period. Leaving the cells in Monocyte Attachment Medium for a longer time or even overnight will induce apoptosis and lead to the loss of the cells.
If necessary, you can reduce the incubation time to 1 hr. In this case, it is advisable to equilibrate the media in the incubator before so that you can immediately and directly add the appropriate amount of PBMC suspension.Related Links and Documents
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When do I need PromoCell’s Monocyte Attachment Medium (C-28051)?
The Monocyte Attachment Medium allows for the efficient adherence selection (within 1 hr) of monocytes from freshly isolated mononuclear cells while maintaining optimal cell health. The Time-consuming and costly immunomagnetic purification of monocytes prior to DC generation is not necessary and can be skipped when using this medium.
.Related Links and Documents
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Which DC medium does PromoCell recommend for freshly isolated MNC or monocytes, which one for cryopreserved monocytes?
The PromoCell DC Generation Media have been developed for the easy and efficient generation of immature as well as fully mature myeloid dendritic cells from peripheral blood monocytes.For freshly isolated mononuclear cells (MNC) and monocytes we recommend our DC Generation Medium DXF (C-28052), for cryopreserved monocytes our DC Generation Medium (C-28050). When using DC Generation Medium (C-28050) with fresh MNC, the Monocyte Attachment Medium (C-28051) is needed in a first step for efficient adherence of the monocyte fraction.
Please see Instruction Manual, Application Note and the graph below for further details.Related Links and Documents
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What are the advantages of monocyte enrichment by adherence selection using your Monocyte Attachment Media (C-28051)?
This method produces large numbers of adherent monocytes in only 1.5 h. If the washing steps are properly performed, 80-90% purity can be expected. The attached cells are “untouched”, since no binding of magnetic microbeads has occured. This also excludes phagocytosis of the microbeads by the monocytes, an event which is unfavorable with regard to cellular health. In addition, the adherence method is time-saving and cost-effective.
Related Links and Documents
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Is it possible to carry out the macrophage differentiation in either a 96- or 384-well plate, so long as the volumes are adjusted accordingly? If not, is it possible for cells to be detached and re-plated in the desired culture plate?
a) We usually perform macrophage differentiation in T75 flasks and 6-well plates. We haven’t tested differentiation in smaller formats. But we assume it will be problematic to thoroughly wash the surface of the wells to remove non-adherent cells after the attachment phase. b) Detachment of the mature macrophages is possible but re-attachment can lead to significant cell loss (30-50%).
Please also keep in mind that working in 96/384 well Format has some inherent drawbacks (e.g. Evaporation of media, dry wells, etc).Related Links and Documents
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I was wondering if you had any suggestions regarding the plasticware to use for monocyte enrichment/macrophage differentiation.
Usually, we do not recommend specific plasticware but we have found that with PromoCell’s Dendritic Cell and Macrophage Generation Media, choice of plasticware can have a lot of an influence. For our Macrophage Generation Media DXF we recommend the Nunc plasticware with Nunclon surface – as not only will the detachment efficiency vary (up to 20%), but also the efficiency of the differentiation process itself may be altered.
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Does PromoCell recommend a specific plasticware to use for the generation of dendritic cells from peripheral blood monocytes?
Usually, we do not recommend specific plasticware but we have found that with PromoCell’s Dendritic Cell and Macrophage Generation Media, choice of plasticware can have a lot of an influence. For our Dendritic Cell Generation Media we recommend to use tissue culture vessels from BD FalconTM.
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Is there is a difference in the percentage of differentiated DCs depending on whether we start with cryopreserved or fresh isolated monocytes?
1)TThere is no significant difference in the percentage of cells that will differentiate into Dendritic Cells. But when using cryopreserved cells, the initial cell loss will be higher compared to when fresh cells are used. i.e. the final number of differentiated cells that can be expected will be higher with fresh cells as a starting material due to lower cell death rate.
Related Links and Documents
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After detaching the macrophages for flow cytometric analysis, I noticed that the washing and centrifugation steps take a long time. Can I shorten the spin time to 10 minutes at 350 x g?
Spinning the cells for 15 min at 350 x g has been proven and tested by PromoCell Research & Development. The QC department uses these settings during testing. Any lower centrifugation value (g-force and/or time) will lead to significant cell loss by means of non-sedimented, but intact, macrophages.
Related Links and Documents
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Why is it not necessary to use Monocyte Attachment Medium (C-28051) when using DC Generation Medium DXF (Ready-to-use, C-28052)?
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Application Notes (4)
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Reference Literature (8)
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Impact of individual intravenous iron preparations on the differentiation of monocytes towards macrophages and dendritic cells
Fell et al.; Nephrol Dial Transplant. 2016 Nov;31(11):1835-1845
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Activated platelets induce an anti-inflammatory response of monocytes/macrophages through cross-regulation of PGE2 and cytokines
Linke et al.; Mediators Inflamm. 2017;2017:1463216
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Selective p38alpha MAP kinase/MAPK14 inhibition in enzymatically-modified LDL-stimulated human monocytes: implications for atherosclerosis
Cheng et al.; FASEB J. 2017 Feb;31(2):674-686
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Biallelic hypomorphic mutations in a linear deubiquitinase define otulipenia, an early-onset autoinflammatory disease
Zhou et al.; Proc Natl Acad Sci U S A. 2016 Sep 6;113(36):10127-32
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TLR4, NOD1 and NOD2 mediate immune recognition of putative newly-identified periodontal pathogens
Marchesan et al.; Mol Oral Microbiol. 2016 Jun;31(3):243-58
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Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease
Zhou et al.; Nat Genet. 2016 Jan;48(1):67-73
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Rhesus macaque theta-defensin RTD-1 inhibits proinflammatory cytokine secretion and gene expression by inhibiting the activation of NF-kappaB and MAPK pathways
Tongaonkar et al.; J Leukoc Biol. 2015 Dec;98(6):1061-70
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Importance of the HIF pathway in cobalt nanoparticle-induced cytotoxicity and inflammation in human macrophages
Nyga et al.; Nanotoxicology. 2015 Feb 13:1-13
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