The combination of PMA and M-CSF induced the differentiation of myeloid-derived monocytes into MDSCs. MDSCs were found to induce immune tolerance by inhibiting the proliferation and activation of T cells, promoting cytokine secretion and inducing T cell transformation to regulatory T cells (T reg).
How long does it take for monocytes to differentiate in PMA?
Flow cytometry analysis confirmed that cells differentiated in 20 ng/mL of PMA had increased levels of monocyte/macrophage markers CD11b and CD14 by 2 and 3 days of incubation, respectively (Fig 5).
What is monocyte differentiation?
Monocyte differentiation. Monocytes are differentiated from the committed precursor termed macrophage-DC precursor (MDP) mainly resident in bone marrow and differentiate into either dendritic cells or macrophages.
Can PMA differentiated THP-1 cells be used as a model for macrophages?
Our goal is to use PMA differentiated THP-1 cells as a model for human monocyte derived macrophages.
How do anti-inflammatory monocytes differentiate into macrophages?
They are highly infiltrative and can be differentiated into inflammatory macrophages, which remove PAMPs and cell debris. In steady state, the patrolling anti-inflammatory monocytes patrol the vasculature to monitor PAMPs and become tissue resident macrophages.
What does PMA do to monocytes?
THP-1 cells are widely used to study monocyte/macrophage function and biology. PMA is often used to stimulate differentiation into cells that mimic human MDMs, in terms of cell morphology, macrophage surface markers, and cytokine production [23].
What does PMA do to macrophages?
PMA at moderate concentrations induced apoptosis in macrophages, and this process appeared to be increased in the presence of myelin. In contrast, microglia were activated by PMA, and greatly increased their phagocytosis of myelin.
What causes monocytes to differentiate?
Monocytes can differentiate into inflammatory or anti-inflammatory subsets. Upon tissue damage or infection, monocytes are rapidly recruited to the tissue, where they can differentiate into tissue macrophages or dendritic cells.
What triggers the differentiation of monocytes into macrophages?
Growth factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and M-CSF play a principal role in their activation: GM-CSF drives the differentiation of “pro-inflammatory” monocytes to M1 macrophages, while M-CSF regulates differentiation of the “anti-inflammatory” subset of monocytes to M0 ...
How does PMA induce macrophage differentiation?
Differentiation into macrophages was induced by incubation of the cells with 200 nM PMA for 24 h. Nuclei were harvested from LPS-stimulated and nonstimulated cells at different intervals, and in vitro transcription of the TNF-α gene was analyzed with specific probes for TNF-α.
What is the function of PMA?
PMA is the most common and potent phorbol ester. It is active at nanomolar concentrations and activates NF-κB in a dose-dependent manner [1]. PMA causes a wide range of effects in cells and tissues and is a very potent mouse skin tumor promoter [2, 3].
How do you differentiate macrophages?
Tissue macrophages can be derived from monocytes. When isolated from blood and cultured in media with serum, adherent monocytes will differentiate into macrophages. For a pure macrophage culture, we recommend that you add factors such as M-CSF.
How do monocytes differ from macrophages in vitro?
Macrophage Differentiation ProtocolIsolate mononuclear cells (day 0). ... Analyze mononuclear cells (day 0). ... Let the monocytes attach (day 0). ... Prepare the complete Macrophage Generation Medium DXF (day 0). ... Wash the adherent cell fraction (day 0). ... Start the macrophage differentiation (day 0).More items...
How can you tell the difference between M1 and M2 macrophages?
M1 macrophages produce nitric oxide (NO) or reactive oxygen intermediates (ROI) to protect against bacteria and viruses. M2 macrophages are alternatively activated by exposure to certain cytokines such as IL-4, IL-10, or IL-13.
How does a monocyte become a macrophage?
Macrophages are formed through the differentiation of monocytes, one of the major groups of white blood cells of the immune system. When there is tissue damage or infection, the monocytes leave the bloodstream and enter the affected tissue or organ and undergo a series of changes to become macrophages.
How do you isolate macrophages?
Allow wash medium to collect in a sterile 50-ml conical centrifuge tube on ice. Centrifuge cells 10 min at 500 × g, room temperature. Discard supernatant. Resuspend cell pellet in macrophage complete medium by tapping tube and pipetting up and down.
Which cell differentiates to a macrophage when exposed to a specific cytokine?
Most macrophages are derived from bone marrow precursor cells that develop into monocytes. These are formed in the bone marrow from stem cells of the granulocytic–monocytic lineage that are exposed to cytokines such as the granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3).
How does LPS affect monocyte differentiation?
Schematic representation of the interrelationship between monocyte differentiation, NF-κB accumulation and translocation, and TNF-α secretion. When exposed to the phorbol ester PMA, THP-1 monocytes differentiate to mature macrophages. During the differentiation process, NF-κB accumulates in the cytoplasm. LPS stimulation induces the translocation of NF-κB into the nucleus, followed by the secretion of TNF-α. In the undifferentiated THP-1 cells, there are only low levels of NF-κB in the cytoplasm, which causes the cells to respond to LPS stimulation in a much slower way and by the secretion of lower levels of TNF-α, compared to the differentiated cells.
How do macrophages contribute to the immune system?
Macrophages play a key role in the orchestration and execution of the innate and adaptive arms of the immune response to bacterial infection. During the infective process, circulating blood monocytes migrate from the vasculature into the extravascular compartment under the influence of many different endogenous and exogenous factors. In the tissues they differentiate to macrophages (2). Upon differentiation, the cell loses its ability to replicate and its antibacterial properties are markedly enhanced, allowing it to participate in the inflammatory and immune responses. The differentiation process is a complex one and is controlled by the expression or activation of several transcription factors (30). However, the events during terminal differentiation of the macrophage leading to its enhanced antibacterial activities are poorly understood.
What is the RPMI for THP-1?
The human monocytic cell line THP-1 (American Type Culture Collection, Manassas, Va.) was maintained in RPMI 1640 media supplemented with 2 mM l-glutamine–100 U of penicillin per ml–100 μg of streptomycin per ml–25 mM HEPES (C-RPMI) and 5% fetal bovine serum (all from Gibco BRL, Gaithersburg, Md.). For the induction of cell differentiation, cells (5 × 105to 106per ml) were seeded in macrophage serum-free medium (macrophage-SFM; Gibco BRL) with 2 to 200 nM PMA for 24 h (29). After incubation, nonattached cells were removed by aspiration, and the adherent cells were washed with C-RPMI three times. THP-1 cells in macrophage SFM with no PMA were used as control (undifferentiated) cells.
What is THP-1 cell?
THP-1 cells are premonocytes, committed to the monocytic cell lineage. They grow in suspension and do not adhere to the plastic surfaces of the culture plates (Fig. (Fig.1A).1A). For the induction of terminal differentiation to macrophage-like cells, THP-1 cells were cultured in the presence of 2, 20, and 200 nM PMA for 4 days. After 20 h of culture with 200 nM PMA, the cells adhered to the dish bottom and had the morphological characteristics of macrophages (Fig. (Fig.1B).1B). Flow cytometry analysis revealed that these cells expressed high levels of CD14, a macrophage-specific differentiation antigen, compared to untreated THP-1 cells. PMA (20 nM) also induced adherence and spreading of the cells after 3 to 4 days of culture. However, a dose of 2 nM PMA was ineffective for the induction of maturation. Based on these results, further experiments using 200 nM PMA for 24 h cell differentiation were conducted.
Popular Answers (1)
To look at macrophages rather than monocytes you would definitely need to treat first with PMA. This treatment will give you "M0" macrophages. Following PMA, LPS/IFNg treatment will give you "M1" macrophages, while treatment with IL-4/IL-13 will polarize to "M2" macrophages.
All Answers (3)
To look at macrophages rather than monocytes you would definitely need to treat first with PMA. This treatment will give you "M0" macrophages. Following PMA, LPS/IFNg treatment will give you "M1" macrophages, while treatment with IL-4/IL-13 will polarize to "M2" macrophages.
Similar questions and discussions
Which conditions do I need to differentiate to THP-1 cell to macrophages?
What is the protocol for collecting peripheral blood monocytes?
Human peripheral blood monocytes, enriched by apheresis, were obtained from the NIH blood bank under protocol 99-CC-0168: “Collection and Distribution of Blood Components from Healthy Donors for In Vitro Research Use.” The protocol was reviewed and approved by the NIH Institutional Review Board (IRB). Signed informed consent was obtained from each donor, acknowledging that his or her donation would be used for research purposes by intramural investigators throughout the National Institutes of Health.
What are the effects of high concentrations of PMA?
Undesirable effects of high concentrations of PMA, have been reported and include activation towards the classically activated M1 state [15,24]. Here we found that differentiation of THP-1 cells in high concentrations of PMA ≥ 50 ng/mL) for 3–4 days results in high sensitivity to LPS resulting in significant cell death. In contrast, cells differentiated in 20 ng/mL of PMA were more resistant to rapid cell death when either exposed to LPS or infected with Salmonella. Furthermore, THP1 cells differentiated at the lower PMA concentration are more bactericidal, than those differentiated with 200 ng/mL PMA, and direct comparison of these cells with human MDMs, differentiated in the absence of activating signals, suggest that they have a similar ability to control intracellular Salmonella. The response of macrophages to Salmonellainfection involves the secretion of cytokines such as TNF-α and IL-12 and chemokines including IL-8 (CXCL8) [6,25–27]. We found that THP-1 cells, differentiated in 20 ng/mL PMA for 3 days, and human MDMs released similar levels of these cytokines, although IL-8 may be released somewhat earlier by the THP-1 cells. Trafficking of Salmonelladuring MDM infection has been shown to involve the acquisition of the late endosomal/lysosomal membrane protein LAMP1 [28]. Our analysis also showed similar kinetics of LAMP1 acquisition in THP1 cells following Salmonellainfection. Interstingly, this THP1 trafficking was similar regardless of the method used for differentiation, suggesting that Salmonellatrafficking in these macrophage like cells is similar to the primary MDMs regardless of their differentiation state.
How are THP-1 cells differentiated?
THP-1 cells were differentiated into macrophage-like cells (THP-1 macrophages) by incubation in the presence of PMA, which leads to a macrophage-like phenotype characterized by changes in morphology and increased cell surface expression of CD11 and CD14 [ 10, 11, 14, 17, 21 ]. Since both the PMA concentration and the duration of treatment affect maturation [ 19] we started by incubating THP-1 cells with PMA (200 ng/mL) either continuously for 1 or 2 days or for 2 days followed by 3 days of rest (5 day). This was based on a report that a minimum concentration of 100 ng/mL (162 nM) should be used for at least 48 hr [ 7 ]. As assessed by light microscopy, changes in cell morphology were dependent on the duration of incubation, with the cells becoming less refractive to light and larger over time ( Fig 1A ). Analysis by flow cytometry showed that surface levels of CD11b and CD14 were lowest in cells differentiated for 1 day and highest in cells differentiated for 5 days ( Fig 1B and 1C ). Since the 2- and 5-day PMA-differentiated cells appeared more macrophage-like than the 1-day cells, we used these conditions for the following experiments.
How long did THP-1 cells stay in the presence of PMA?
Supernatants were collected from uninfected and Salmonella infected primary human macrophages and THP-1 cells plated for 3 days in the presence of 20 ng/mL PMA at 6 h and 24 h pi. Cytokine secretion was measured by ELISA per the manufacturer instructions (R&D Systems).
What is THP-1 cell?
THP-1 cells are widely used to study monocyte/macrophage function and biology. PMA is often used to stimulate differentiation into cells that mimic human MDMs, in terms of cell morphology, macrophage surface markers, and cytokine production [ 23 ]. However, the amount of, and duration of incubation with, PMA varies widely.
How long to incubate Salmonella cells?
(E. coliculture conditions were identical to those used for Salmonellagrowth). Where indicated cells were treated with LPS from Salmonella entericaserotype Minnesota (10 ng/mL, Sigma) for 30 min. For propidium iodide assays, cells were incubated with propidium iodide (1 μg/mL, Life Technologies) for 15 minutes at the appropriate time post-infection and immediately analyzed by fluorescence microscopy without fixation. For each sample, several fields from duplicate wells were measured. For DAPI staining cells were fixed in 2.5% PFA for 10 min at 37°C before staining with DAPI (Life Technologies) for 10 min at RT and mounting in Mowiol (Aldrich).
What was used for FLICA assay?
For FLICA assays, control cells were treated with either vehicle (DMSO) or 1 μM staurosporine (Sigma) for 3 h prior to analysis. Cells were also incubated with Salmonella, E. colior LPS. Cells were then incubated with poly-caspase inhibitor reagent FAM-VAD-FMK (Immunochemistry Technologies) for 1 h followed by a 10 min wash. Cells were immediately analyzed with several fields from duplicate wells being measured for all conditions.