Pma differentiation protocol. After differentiation, the cells present .
Pma differentiation protocol. Immunol. Nov 18, 2022 · In this study, we used a standardized differentiation protocol using PMA and compared the response of macrophages derived from THP-1 and U937 cells to M1-and M2-polarizing conditions. Despite this understanding, the absence of a standardised protocol has limited the use of differentiated SH-SY5Y cells in high-throughput assay formats. BMDM are primary cells and have the biological function and properties of macrophages differentiated from monocytes because there are mature, in contrast to macrophage cell lines, which are immature. T lymphocytes are orchestrators of adaptive immunity. Optimization of macrophage differentiation protocol for THP-1 monocytes. There is an article though that made an effort to optimize the PMA differentiation protocol of THP-1 cells so that they would as closely as possible resemble primary human monocyte-derived What would be a good protocol to differentiate U937 cells with PMA in 24 well plates? U937 concentration PMA concentration and length of treatment Phorbol 12-myristate 13-acetate (PMA) induces differentiation of human leukemic HL-60 cells into cells with macrophage-like characteristics and enhances the susceptibility of HL-60 cells to the Helicobacter pylori VacA toxin (de Bernard, M. Macrophages were differentiated starting from the human monocytic cell line THP-1. Phagocytosis was subsequently assessed by incubating cells with fluorescent zymosan particles (green). Thus, another goal of the present systematic review was to provide a comprehensive summary of methods of THP-1 cells differentiation to ascertain the best possible polarization procedure. , M. The cells were photographed at ϫ 200 magnification with a phase Nov 1, 2022 · Despite the abundant use of THP-1 cells in macrophage studies, there are no standardized protocols for reliable THP-1 differentiation into macrophages. , 2010), especially THP-1-differentiation with PMA which has been demonstrated to result in highly variable outcomes between laboratories (Xia et al. , and Montecucco, C. 2%) and cytokine secretion and midkine responses were the important discriminators of the level of macrophage differentiation. Abstract THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) are widely used as a model for function and biology of human macrophages. This is because, following differentiation using phorbol 12-myristate 13-acetate (PMA), THP-1 cells acquire a macrophage-like phenotype, which mimics, in many respects, primary human macrophages. This rest period ensures optimal differentiation of THP-1 cells into terminally differentiated macrophages. In this study, the induction were optimized by evaluating the differentiation effects of mac-rophages under three conditions which are different PMA concentrations, different induction times and different cell seeding densities, and the induction effect using the above optimized protocols was confirmed by optical microscope, flow cytometry and Aug 19, 2021 · Here, we provide an improved culture protocol for immature naïve mouse CD4 + T cells, including details for splenocyte isolation, naïve CD4 + T-cell purification and differentiation, and functional evaluation via flow cytometry. , Rappuoli, R. The readout was examined using SEAP expression in differentiated THP1-XBlue™ cells cultured in 18% versus 5% O 2 in the absence and presence of LPS. For the latter cell types, we recommend using Method 2, the long-term Th17 cell polarization culture protocol. , 2018) was confirmed (Fig. Here, we provide a dataset for validated protocol when THP-1 cells are differentiated into LPS inducible macrophages by using phorbol-12-myristate-13-acetate (PMA). Despite the widespread use of THP-1 cells in studies elucidating macrophage responses to Stimulation of human monocyte derived macrophages with PMA Phagocytosis assay using PMA-differentiated THP-1 cells THP-1 cells (immortalised human monocyte model) were treated with PMA to induce macrophage like characteristics. Although size is not a usual parameter for measuring differentiation, it appears to be an indicator of differentiation in proliferating cells. without the differentiation induced by phorbol 12-myristate 13-acetate (PMA) by bacillus Calmette-Guérin (BCG). For my protocol I dilute my cells to 0. However, this concentration might be high enough to upregulate the expressions of some genes in differentiated macrophages, which could overwhelm gene expression increases induced by other stimuli. Jun 15, 2023 · The cells can be fixed, permeabilized, and stained for different markers at this point. The cells exhibit phagocytic activity and responsiveness to chemotactic stimuli. Apr 16, 2025 · To substantiate the macrophage-like characteristics acquisition for the newly transitioned state after 48 h of PMA treatment towards THP-1 cells, RT-qPCR was run immediately post-PMA treatment to determine the relative fold change of the macrophage-specific differentiation antigen CD14 surface marker gene. Feb 17, 2023 · According to both the HCL and Venn diagrams of co-expression, the greatest differences between M1 and M2 macrophages were in Protocol A. J. 5%), actinomycin D, and retinoic acid. This protocol uses adequate resting periods and a prolonged polarization process to The phorbol 12-myristate 13-acetate (PMA)-induced U937 cell line has been widely used as an in vitro model for studying the functions of human macrophages. Let the monocytes Macrophage Differentiation from freshly isolated PBMC This protocol describes the in vitro dif-ferentiation of M1 or M2 macrophages directly from freshly isolated peripheral blood mononuclear cells (PBMC) using the Macrophage Generation Media to induce differentiation. The differentiation of THP-1 monocytes into macrophages is mainly conducted at a phorbol 12-myristate 13-acetate (PMA) concentration of 10-400 ng/ml. Mar 13, 2023 · A key feature of this protocol is the utilisation of three distinct culture stages (PMA priming, rest, and cytokine differentiation) to generate cultures enriched for M0, M1 and M2 macrophages (Supplementary Figure 1A). We used one defined protocol to allow for comparisons between the biological response of the monocytic cell lines. Upon stimulation, differentiated THP-1 cells will adhere to the plate and secrete upregulate cytokines. Therefore, we performed a CRISPR screen in monocytic Thp1 cells, differentiated into macrophages using PMA, using CD14 as a readout. Using a manual multi-channel Thus, the use of different PMA differentiation protocols introduces the likelihood of naively and incorrectly comparing the response of presumed uniform, yet functionally different, macrophage Jun 21, 2021 · In this study, we employed a dimethyl labeling-based quantitative proteomics approach to determine the changes in the protein repertoire of macrophage-like cells differentiated from THP-1 monocytes by three commonly used PMA-based differentiation protocols. The cells are phagocytic (for both latex beads and sensitized erythrocytes) and lack surface and cytoplasmic immunoglobulin (www. (A) Primary human CD14 + monocytes or (B) THP-1 monocytes were stimulated with GM-CSF (5 ng/ml) or PMA (100 ng/ml Jul 29, 2015 · We herein investigated the role of NRF2 in macrophage differentiation using the human monocytic U937 cell line and phorbol-12-myristate-13-acetate (PMA). May 15, 2018 · Bone marrow-derived macrophages (BMDM) are primary macrophages obtained by in vitro differentiation of bone marrow cells in the presence of macrophage colony-stimulating factor (M-CSF or CSF1). , et al. Oct 7, 2020 · CD14 is a co-receptor for bacterial LPS, and can potentiate inflammatory signalling when upregulated on myeloid cells during differentiation. Experiments using THP-1 and monocytic cell line HL-60 have revealed that PMA induces a greater degree of differentiation than Vit D 3 [51]. [113] used macrophage-derived PMA-differentiated THP1-XBlue™ cells to investigate whether oxygen tension influences THP-1 cell differentiation and primary macrophage functions. This protocol is a guide to differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. I would appreciate any advice. e. Increased adhesion of PMA-induced megakaryocytes was also observed on differentiation day 5 compared to K562 or DMSO-treated cells (Figure S1C in Supplementary Materials). Nov 12, 2020 · Differentiation therapy has been successfully applied clinically in cases of acute promyelocytic leukemia (APL), but few differentiation-induction agents other than all-trans retinoic acid (ATRA Jan 15, 2014 · Previously we have published a protocol for transfection of THP-1 macrophages, which in brief pre-differentiates THP-1 monocytes for 48h using 100ng/ml PMA, followed by detachment of the cells and eletroporation using Lonza nucleofector technology and finally includes further 48h of differentiation with 100ng/ml PMA. THP-1 cells were treated with 10 ng/mL PMA or MCSF as described in materials and methods and CD14 expression determined by Jun 7, 2022 · Even though SH-SY5Y cells are widely explored, a complete description of the resulting proteomes and cellular reorganisation during differentiation is still missing. As a conclusion the results have shown that a modified PMA differentiation protocol (20 ng/ml and 48 hours incubation) might enhance macrophage differentiation of THP-1 cells without induced cell death (viability 92. Parental THP-1 and freshly thawed ThawReady™ THP-1 cells were plated and treated with PMA for 3 days to stimulate differentiation into macrophage-like cells. May 19, 2023 · After differentiation, all THP-1m cells were washed out of the PMA-containing medium and cultured in fresh CGM without PMA for 21 days. 5. Add ~150,000 undifferentiated THP1 cells to each well (~150 uL of a dense culture). The induced cells show growth inhibition, morphological changes to monocyte-macrophage-like cells, and increases in nitrobl … In the present study, we highlighted how AKT signaling appears as a vital regulator of differentiation, survival and surface expression of CD14, CD206 and CD83 in PMA-induced U937 cell differentiation to macrophage-like cells. On day 7 of differentiation, PMA-induced megakaryocytes were more adherent to the culture plate and were more spread out than K562 or DMSO-treated cells (Figure 1 C). (A) Comparison of flow cytometric cell features between THP-1 monocytes upon treatment with PMA by zebra plots displaying side scatter area (SSC-A) on the y-axis against forward scatter area (FSC-A) on the x-axis. Macrophage Differentiation Protocol Isolate mononuclear cells (day 0). Figure 4: Increased mRNA expression of CD14 and CD36 in PMA induced macrophage-like cells. Dimethyl labeling-based quantitative proteomic approach was employed to identify and quantify the proteome changes in Condition A (Light), Condition B (Medium), and Condition C (Heavy). A) Summary of the protocols used to differentiate premonocytic THP-1 cells into resting (M0; PMA), classically activated (M1; LPS+IFN-γ), and alternatively activated (M2; IL-4+IL-13) macrophages. Also, the expression of microRNA-155 (miR-155) and miR-125b in PMA-treated human monocytic cell line has not Mar 30, 2016 · Figure 1. Analyze mononuclear cells (day 0). 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 ). The findings indicate that a change in the balance of the cellular mechanisms employed for nucleoside transport occurs during the monocytic differentiation of HL-60 leukaemia cells. We would like to show you a description here but the site won’t allow us. The data were Jul 4, 2024 · Despite the wide use of this model, differentiation protocols using phorbol 12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D 3 (1,25D 3) vary drastically between studies. g. Both ATRA- and DMSO-induced dHL-60 cells were able to release NETs upon PMA and CI stimulation; dHL-60 cells in serum-free medium produced more NETs than those in serum-containing medium. Using small molecules to direct the differentiation of iPSCs into MNs Aug 24, 2017 · Treatment of THP-1 monocytes with PMA ceased cell proliferation and induced differentiation into adherent CD11b and CD11c double positive resting macrophages referred to as M0 (Fig 1A and 1B). Phorbol 12-myristate 13-acetate (PMA), also known as 12-O-tetradecanoylphorbol-13-acetate or TPA, is a naturally occurring phorbol ester found in plants like croton oil. Differentiated cells are then challenged with lipopolysaccharide (LPS) to stimulate the inflammatory cascade. (1998) FEBS Lett. This finding suggests that THP-1 cells are well differentiated by 5 ng/ml PMA, and that the resulting differentiated macrophages respond well to secondary weak stimuli without being overwhelmed by undesirable gene upregulation induced by PMA. Scheurlen Mar 15, 2024 · We then describe detailed procedures for freezing/thawing of monocytes and differentiation into polarized M1 and M2 macrophages. Isolate fresh PBMCs from buffy coats using your routine protocol. PMA differentiated THP-1 cells are the main cells I work with - what concentration of PMA are you using and how long are you allowing cells to differentiate? I allow 72 hours for cells to differentiate in PMA treated media, then wash and change to normal media and allow the cells to rest overnight and get about 90% adherence and differentiation. This protocol is not intended as a culture method for generating differentiated Th17-like cells or cloning of Th17 cells. Abstract THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) are widely used as a model for function and biology of human macrophages. The most common protocol involved 48h culture with 150 nM PMA, after which cells were used either immediately or after 1-2 days rest in PMA-free medium. The immortalized and proliferative cell line SH-SY5Y is one of the most commonly used cell lines in neuroscience and neuroblastoma research. THP-1 monocytes can be differentiated to macrophages by incubation with phorbol-12-myristate 13-acetate (PMA), These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells. The monoblast-like human histiocytic lymphoma cell line U937 can be induced by phorbol 12-myristate 13-acetate (PMA) at concentrations of 8-16 ng/ml to undergo differentiation. However, undifferentiated SH-SY5Y cells share few properties with mature neurons. NO. Initial studies were done utilizing a long-term THP-1 culture, grown at a higher density (Condition I). Apr 22, 2024 · Agents used for differentiation of THP-1 cells include phorbol 12-myristate 13-acetate (PMA), vitamin D, and macrophage colony-stimulation factor M-CSF (Chanput et al. , 2014). Flow Cytometric Analysis, single and Multi-analyte Assay of Cell Supernatants. A key feature of this protocol is the utilisation of three distinct culture stages (PMA priming, rest, and cytokine differentiation) to generate cultures enriched for M0, M1 and M2 macrophages ( Supplementary Figure 1A ). We analyzed mRNA expression in un-induced, hemin-induced and PMA-induced K562 cells by differential display reverse transcription polymerase chain reaction (DDRT I'm currently preparing a PMA (phorbol 12-myristate 13-acetate) stock solution that will be used to differentiate THP-1 cells. 2. Differentiated THP-1 cells acquire macrophage-like characteristics including more adherence and altered cell function. This protocol constitutes an optimized version of a previously published protocol by our group. In the presence of PMA, most THP-1 cells attached to the bottom of the Petri dish and differentiated into macrophages ( Fig. Download scientific diagram | Differentiation of THP-1 cells to generate macrophages. Aug 12, 2023 · These findings suggest that UHRF1 and DNMT1 play roles in regulating PMA-induced differentiation of THP-1 cells by regulating transcription and DNA methylation of differentiation-related genes. Mar 14, 2018 · THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) are widely used as a model for function and biology of human macrophages. The lack of a standardized protocol has resulted in THP-1–derived macrophage populations that are inconsistent and difer Jan 15, 2014 · Previously we have published a protocol for transfection of THP-1 macrophages, which in brief pre-differentiates THP-1 monocytes for 48 h using 100 ng/ml PMA, followed by detachment of the cells and eletroporation using Lonza nucleofector technology and finally includes further 48 h of differentiation with 100 ng/ml PMA. Abstract In order to investigate the effects of RhoA/ROCK signaling in macrophage differentiation, we used 100 ng/mL PMA to induce macrophage differentiation from U937 cells in vitro. THP1 cells are a monocytic cell line which can be differentiated to a macrophage like phenotype using phorbol 12-myristate 13-acetate (PMA). (A) Flow cytometry analyzed the expression of CD68 by THP-1 cells and M0 macrophages. K562 cell line has been used as a model of common progenitor of erythroblasts and magakaryocytes and can be differentiated into erythroid and megakaryocytic lineages by hemin and phorbol myristate acetate (PMA) respectively. , [10] highlight that, for THP-1 differentiation to occur, PMA exposure should range from 24 Differentiation of the SH-SY5Y cells is a necessary step to obtain cells that express mature neuronal markers. , Moschioni. qPCR was performed to quantify (A) CD14 and (B) CD36 mRNA expression. Dec 6, 2020 · Does anybody use PMA to differentiate HL60 cells? If so, I would like to know your protocol. These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells. Mar 1, 2020 · Our efforts to refine and standardize THP-1 differentiation and polarization protocols were based on sequential optimization of each stage in the protocol. In this study, we present an optimized neuronal differentiation protocol for SH-SY5Y that requires only two work steps and 6 days. Jan 15, 2014 · Previously we have published a protocol for transfection of THP-1 macrophages, which in brief pre-differentiates THP-1 monocytes for 48 h using 100 ng/ml PMA, followed by detachment of the cells and eletroporation using Lonza nucleofector technology and finally includes further 48 h of differentiation with 100 ng/ml PMA. Jan 1, 2018 · Differentiation Protocol for SHSY5Y This protocol allows to differentiate SHSY5Y gliablastoma cells to neuron-like cells. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced phorbol-12-myristate-13-acetate (PMA)-differentiated lipid deposition in human monocyte (THP-1) macrophage model to observe cell death. Furthermore, PMA promoted U937 cell accumulation in the G2M phase of the cell cycle during differentiation. Count and analyze the isolated PBMCs for monocyte content, (e. Neutrophil extracellular traps (NET) induced by different stimuli: A comparative proteomic analysis. Both ATRA‑ and DMSO‑induced dHL‑60 cells were able to release NETs upon PMA and CI stimulation; dHL‑60 cells in serum‑free medium produced more NETs than those in serum‑containing medium. This is an insoluble (in water) phorbol ester that must be resuspen Understanding how immune cells such as macrophages interact with cancer cells is of increasing interest, as cancer treatments move towards combining both targeted- and immuno- therapies in new treatment regimes. ) vary widely from lab to lab. May 1, 2021 · THP-1 cells, a human leukemia monocytic cell line, differentiated by phorbol myristate acetate (PMA) are widely used as surrogate of human macrophages… The differentiation of THP-1 monocytes into macrophages is mainly conducted at a phorbol 12-myristate 13-acetate (PMA) concentration of 10-400 ng/ml. Jan 13, 2021 · The differentiation of THP-1 monocytes into macrophage-like cells (M0) by PMA is a common method, whereas studies about the differentiation into dendritic-like cells are relatively limited (56). Apr 2, 2009 · Inevitably, it would require tight regulation. We use flow cytometry to analyze Annexin V and 7-AAD double staining to detect cell death, using the whole cell and employing scanning electron microscopy to observe the cell membrane morphology. Culturing and differentiation protocol: This document contains detailed and comprehensive information about cell culture media, subculturing, freezing and thawing, and differentiation protocols of these cells. Firstly, we applied reduced concentrations of PMA to prime the THP-1 cells for differentiation. the cells react differently to differentiation substances depending on the cell density Liu et al. Sep 1, 2018 · Furthermore, low expression of CD14/TLR2 and high expression of CD68/CD86 were observed. Centrifuge the cell suspension (350 x g, 4 minutes) and resuspend the cell pellet in RPMI 1640 + 10% FBS + 5 ng/mL PMA + 0. Methods 430, 64-70 (2016). L. 5x106 cells per ml (or well) and add PMA at a concentration of Hello Dayoon I used various protocols for the optimization of THP-1 differentiation into the macrophages (M0). Protocol for the Differentiation and Characterization of Human Th2 Cells. In this study, we found that mitochondrial functions changed obviously when K562 cells were induced to megakaryocytic differentiation by phorbol 12-myristate 13-acetate (PMA). I seed 2 X 10^6 cells in each well in a 12-well-plate, and I tried 10, 50, 100, 150, 200 ng/mL PMA for 48hr, but the differentiation efficiency is too low that I am unable to do the downstream Sep 17, 2021 · Determining the antigen specificities of the endogenous T-cell repertoire is important for screening naturally occurring or therapy-induced T-cell immunity and may help identify novel targets for T-cell-based therapies. Cells were metabolically labeled (SILAC) and differentiation was induced by addition of phorbol-12-myristate 13-acetate (PMA) for 3 days followed by a period of resting for 1 day in PMA-free medium. Cell differentiation, phagocytosis and calcium influx were analyzed by flow cytometry. Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line - a 2 minute Preview of the Experimental Protocol Katharina M. Published protocols using the promonocytic cell line THP-1 have tended to result in cells that closely resemble classically-activated macrophage … Apr 12, 2024 · Figure: THP1 cell differentiation into macrophages using PMA. : HY-18739, MCE) to induce adherent differentiation, but how to do it specifically? I want to ask for a detailed protocol, including: We would like to show you a description here but the site won’t allow us. Induction of differentiation in THP-1 cells by PMA. The conditions were; Harvest THP-1 cells and perform a cell count (e. Introduction cell line (ATCC: TIB-202) is a human monocytic cell line derived from the peripheral blood of a one year old human male with acute monocytic leukemia (Tsuchiya S. org). In conclusion, our results indicate a synergistic effect between PMA and VD3 in U937 cells differentiation into both monocytes or macrophages and we propose a modified PMA differentiation protocol to enhance monocyte/macrophage differentiation of U937 cells. It describes a procedure for transfecting premature THP-1 macrophages, which subsequently can be further differentiated into mature macrophages by PMA without a loss of cell viability. the cells react differently to differentiation substances depending on the cell density (Liu et al. Importantly, this protocol enabled us to investigate at which stage/s in macrophage differentiation PHX might act. Aug 8, 2015 · Monocyte differentiation into macrophages Human THP-1 monocytes were differentiated into macrophages by an incubation in the presence of phorbol 12-myristate 13-acetate (PMA). The present study was performed to Classical Macrophage Differentiation Place sterilized 18 mm diameter circular coverslips into the well of a 12-well plate. May 3, 2024 · Abstract Cell death is a fundamental process in all living organisms. The phorbol 12-myristate 13-acetate (PMA)-induced U937 cell line has been widely used as an in vitro model for studying the functions of human macrophages. The cells are easy to culture and These data indicate that exposure of monocytic THP-1 cells to 25 nM PMA over 48 h, followed by a recovery period of 24h in culture in the absence of PMA, was the optimal protocol for the differentiation of THP-1 cells. The small peak observed over the neutrophil cell type in the PMA treated cells is due to the fact that DMSO is the solvent for PMA, potentially playing a role in the differentiation process. When Jurkat cells are stimulated by PMA and a co-stimulator, such as PHA, IL-2 production is strongly enhanced2. May 9, 2014 · Mitochondria are involved in the regulation of cell differentiation processes, but its function changes and molecular mechanisms are not yet clear. Jun 21, 2021 · Abstract Macrophages are sentinels of the innate immune system, and the human monocytic cell line THP-1 is one of the widely used in vitro models to study inflammatory processes and immune responses. at are inconsistent and difer significantly in terms of phenotype and function. (A) The experimental strategy employed for comparative proteome analysis in response to varying PMA-mediated differentiation protocols. The protocol presented herein is routinely used for culturing and differentiating THP1 cells with PMA. The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus. Here we compare several differentiation conditions and compare the ability of THP-1 macrophages to interact These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells. Jan 27, 2016 · These data indicate that exposure of monocytic THP-1 cells to 25 nM PMA over 48 h, followed by a recovery period of 24h in culture in the absence of PMA, was the optimal protocol for the differentiation of THP-1 cells. I have gotten good results with this. Jan 17, 2018 · PPM1A is kinetically upregulated during monocyte-to-macrophage differentiation. (A) Following treatment with PMA at the indicated concentrations for 24 h, THP-1 cells adhered My protocol is treat with PMA 10 ng/mL for 2 days, followed by washing with sterile warm dPBS 2x, followed by 2 days of resting before experiments. After differentiation, the cells present Induction of HL-60 cells for five days produced the best results in terms of differentiation markers and cell viability. In the present study, a reliable human osteoclast model was established using the THP‑1 cell line. Therefore, we decided to compare different polarization protocols from the perspective of the transcriptome of M1/M2 macrophages polarized according to these protocols. THP-1 cells were incubated for 24 h without (A) or with (B) PMA (200 nM). I did also this thing: PMA differentiation 1d->recovery 1d->PMA 10ng/ml treatment for 0,5h and 20h and it looks promissing but I got strangely low number of cells (also your problem),event As the use of a variety of PMA differentiation protocols likely generates a range of functionally distinct macrophage subsets, clinical applicability and direct comparisons of in vitro studies are compromised. THP-1 cells can be differentiated into macrophage-like cells Jun 3, 2018 · Helly, I've currently bought 1 mg of PMA from sigma to treat U937 cell line differentiation, but now, I'm a bit confused with how to prepare it. Grodzki et al. Mar 1, 2016 · Collectively, the data of the current study demonstrate that the differentiation of THP-1 cells, using different PMA differentiation protocols, generates macrophages at various stages of maturation. Petretto, A. Results: Clearly, PMA induced THP-1 differentiation occurs in adherent culture conditions more than nonadherent culture conditions by increasing CD11b expression up to 90%, which was not affected by PD98059 when cells were exposed to PMA first (post-PD) but inhibited when PD98059 was treated prior to PMA treatment (pre-PD). Here, we relatively quantify the proteomes of cells grown under standard conditions and obtained from two differentiation protocols employing RA or a combination of RA and PMA. Using flow cytometry, Domainex confirmed successful differentiation of cells, using forward scatter (FSC) vs side scatter (SSC) plots, and histograms showing expression levels of different surface markers. , Telford, J. Methods: A morphological and quantitative analysis of markers related to dopaminergic and cholinergic neurons, but also other phenotypes, was performed. I culture in 1640 RPMI Accordingly, we compared the function and phenotype of THP-1 macrophages generated using the range of published PMA differentiation protocols, specifically in response to the pro-inflammatory stimulus, lipopolysaccharide (LPS). Nitric oxide (NO), an intracellular messenger, is critical in regulating cell differentiation. Replace the media with fresh RPMI without PMA for 24 h. This protocol provides foundation to study iPSC monocytes and their progenies such as macrophages, microglial, and dendritic cells. Phorbol 12-myristate 13-acetate (PMA) is commonly used to activate protein kinase C (PKC) and a Standard Reagent for THP1 cell differentiation into macrophages-type cells. Here, we provide an optimized protocol that can be used to diferentiate ATCC Assay Ready 3. responsive after differentiation. Abstract K562 cell line has been used as a model of common progenitor of erythroblasts and magakaryocytes and can be differentiated into erythroid and megakaryocytic lineages by hemin and phorbol myristate acetate (PMA) respectively. However, this concentration might be high Grodzki et al. Apr 1, 2021 · There is a variety of differentiation protocols for these monocytic cells into a macrophage-like phenotype by using different stimulation factors [2]. 1. After washing the PMA-differentiated cells twice, fresh media was added, and the cultures were incubated for an additional 18 h at 37°C in 5% CO2 humidified atmosphere before the addition of extracts. These cells express some macrophage differentiation markers (CD11 , CD14 and CD36) and can phagocytose and release TNF to a greater extent than untreated cells 11. , 2013). Aug 2, 2021 · This protocol is a guide to differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. If you want to mimic primary human macrophages as closely as possible, the following open access paper thoroughly compares different THP-1 differentiation protocols to primary cells THP-1 is a human monocytic cell line originally isolated from a 1-year old male subject with acute monocytic leukemia (Tsuchiya et al. Also U937 pro-monocytes differentiate into mature monocytes or into macrophages upon PMA-treatment. In this research, we examined the differentiation effect of three Differentiation of THP-1 and U937 Monocytes into Macrophages As already mentioned before, nearly all THP-1 cells start to adhere to culture plates and differentiate into macrophages after exposure to PMA. The protocol for the multiparametric inflammation assay is given below. et al. There are many different protocols for THP-1 differentiation to macrophages using PMA. Given that differences in differentiation protocols have the potential to impact The combination of PMA and M-CSF induced the differentiation of myeloid-derived monocytes into MDSCs. Increases in cytokine secretion from stimulation with PMA and LPS are shown in Figure 3. There is a variety of differentiation protocols for these monocytic cells into a macrophage-like phenotype by using different stimulation factors [2]. Dec 1, 2022 · In this study, we used a standardized differentiation protocol using PMA and compared the response of macrophages derived from THP-1 and U937 cells to M1-and M2-polarizing conditions. Scale bar for 10×: 500 μm; 20×: 100 μm; fluorescence image: 100 μm. Cytokines released by THP-1 cells and PMA-differentiated THP-1 cells increased in a dose-dependent manner at each time point. To identify genes and pathways involved in differentiation of Jurkat T PMA alone, or a combination of RANKL and MCSF alone, is insufficient to stimulate THP‑1 cell differentiation into osteoclasts. Dec 2, 2016 · Thrombomodulin was associated with PMA-induced monocytic THP-1 cell differentiation TM is expressed on the surface of monocytes and is important for the regulation of THP-1 cell migration 27. Akt1-phosphorylated Serine 473, Bad-phosphorylated Serine 136 and Caspase9-phosphorylated Serine 196 were tested by flow cytometry for the involvement of the Akt1 signaling pathway during differentiation in addition to the expression of CD14, CD206 and CD83. However, the concentrations and Download scientific diagram | PMA induced THP-1 cells differentiation into macrophages (A,B) THP-1 cells develop characteristic macrophage morphology after exposure to PMA. As demonstrated, increased neutrophils can be observed with DMSO treatment, and increased monocytes are observed with PMA treatment. However, the concentrations and duration of PMA treatment vary widely in the published literature and their effect on protein expression is not fully deciphered. DNA cell Feb 5, 2020 · Treatment conditions would depend upon the question you are planning to ask using PMA differentiated THP-1 cells that are stimulated by LPS. Naive T cells can be differentiated into specific T cell subsets in in vitro culture in response to specific cytokine stimulation. Here, we review reported differentiation protocols for obtaining spinal MNs, summarize the advantages and disadvantages of each strategy, and discuss possible solutions for the remaining challenges. Procedural Notes Method 1 is useful as a rapid test for potential IL-17 production by activated lymphoid cells. This protocol uses adequate resting periods and a prolonged polarization process to create a distinct M2-like macrophage subtype. I have 1 mg of PMA and the supplier (ThermoFisher) states to dissolve this in 1 mL of DMSO for a solution of 1 mg/mL. , Bruschi, M. , 1980). , Papini, E. 34 differentiated from THP-1 monocytes by three commonly used PMA-based differentiation 35 protocols. Subsequently resuspend the cells at 100X10 6 PBMCs/ml in Monocyte Attachment Medium (C-28051). using the FSC/SSC plot of a flow cytometer). HL-60 cells were differentiated with all-trans retinoic acid (ATRA), dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) and stimulated with phorbol 12-myristate 13-acetate (PMA) or calcium ionophore A23187 (CI). 436, 218–222). Dec 13, 2019 · To induce differentiation into the macrophage-like phenotype, monocytes at a density of 0. A 24 h incubation in the presence of 150 nM PMA followed by 24 h in control medium was finally selected as differentiation protocol. I would like to know is someone have a good protocol and/or experience to differentiate the THP-1 cells into osteoclasts. Aug 2, 2021 · Anti-inflammatory tumor associated macrophages are linked to progression and poor prognosis in cancer. Exposure Time Chanput et al. We examined the mechanism by which HL-60 After differentiation, all THP-1m cells were washed out of the PMA-containing medium and cultured in fresh CGM without PMA for 21 days. , 2023. Oct 4, 2024 · This protocol outlines the differentiation of ATCC Assay Ready THP-1 monocytes (ATCC® TIB-202-AR™) into macrophage-like cells using PMA, eliminating the need for prior cell culturing. (A) Experimental outline for comparative kinome profiling of undifferentiated (monocytic) and differentiated (macrophage-like) THP-1 cells. PMA, through its activation of PKC, can activate T-cells and stimulate low-level production of IL-2. PMA is a potent activator of Protein Kinase C, which in turn activates NF-kB in vitro. Protocol for the Differentiation and Characterization of Human Th17 Cells. Once differentiated (M0 macrophages), they were incubated with IL-4 and IL-13 in order to obtain M2 polarized macrophages or with IFN-gamma and LPS for classical macrophage activation Discussion The protocol described herein details a method to produce large numbers of BMDMs. Learn how to efficiently generate monocyte derived macrophages with Thermo Fisher Scientific’s detailed in vitro protocol for macrophage differentiation using M-CSF. Few studies on the cytotoxic May 13, 2024 · This cell line is sensitive to culture conditions; e. Diferentiation Media Preparation: Add PMA to the complete media at a working final concentration of 100 ng/mL for this assay Mar 14, 2018 · Abstract THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) are widely used as a model for function and biology of human macrophages. However, this concentration might be high Aug 8, 2015 · Methods: We set up a model of macrophage polarization, starting from THP-1 monocytes differentiated into macrophages using PMA (Phorbol 12-myristate 13-acetate). This protocol can also be applied for immature human CD4 + T cells. Here, we describe a rapid, sensitive, and high-throughput protocol for expanding antigen-specific T cells from human peripheral blood mononuclear cells in vitro following May 25, 2022 · Differential susceptibility to lipopolysaccharide affects the activation of toll-like-receptor 4 signaling in THP-1 cells and PMA-differentiated THP-1 cells We tested the response of THP-1 cells to the common macrophage differentiation factors PMA and M-CSF as described in materials and methods to evaluate whether PMA or M-CSF was able to induce expression of CD14, a marker of monocyte lineage. May 1, 2021 · THP-1 cells, a human leukemia monocytic cell line, differentiated by phorbol myristate acetate (PMA) are widely used as surrogate of human macrophages… Is there a protocol for the differentiation of THP1 cells? Treatment with phorbol myristate acetate (PMA, catalog #tlrl-pma) induces differentiation of THP1 cells into adherent macrophage-like cells. Although PMA is a commonly used agent for in vitro macrophage diferentiation, the conditions used (PMA concentration, length of treatment, etc. May 3, 2024 · This protocol is based on LPS and ATP-induced death of PMA-differentiated THP-1 macrophages. In order to demonstrate successful differentiation leading to activated macrophages, mRNA accumulation of TNFalpha and IFNbeta was analyzed by real-time PCR. Apr 20, 2024 · In contrast to macrophages, the responses of monocytes infected with the mycobacteria have been less investigated. atcc. The protocol presented herein is routinely used for culturing and differentiating THP-1 cells with PMA. Feb 28, 2020 · Several monocyte-to-macrophage differentiation protocols exist, with phorbol 12-myristate-13-acetate (PMA) being the widely used and accepted method. PMA is commonly used to activate protein kinase C (PKC), a family of enzymes involved in various cellular processes such as cell growth, differentiation Mar 1, 2020 · PMA concentration is critical for adherence, marker gene transcription and M (IL-4) polarization: Higher PMA concentrations inhibit differentiation of THP-1 to cells transcribing M (IL-4) markers Feb 22, 2018 · Convenience versus Biological Significance: Are PMA-Differentiated THP-1 Cells a Reliable Substitute for Blood-Derived Macrophages When Studying in Vitro Polarization? The addition of PMA alone results in cells attaching to tissue culture plastic and adopting a stellate morphology. Oct 23, 2023 · PMA was utilized to stimulate the differentiation of the U937 cell line into macrophage-like cells at a concentration of 10 nM. Add sufficient medium to bring the well volume up to 1 mL. , Pratesi, F. Jan 13, 2010 · These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells. Our analysis shows that variations in PMA concentration and duration of rest post- 36 stimulation result in downstream differences in the protein expression and cellular processes. To identify genes and pathways involved in differentiation of Jurkat T cells towards Th1 and Th2 subtypes we performed comprehensive transcriptome analyses of Jurkat T cells stimulated with various stimuli Feb 27, 2020 · Several monocyte-to-macrophage differentiation protocols exist, with phorbol 12-myristate-13-acetate (PMA) being the widely used and accepted method. Given that differences in differentiation protocols have the potential to impact THP-1 morphology after differentiation I am working with differentiated THP-1 cells, using 25 ng/mL PMA during 24h, followed by 48h resting with PMA-free media. In this study, the induction were optimized by evaluating the differentiation effects of macrophages under three conditions which are different PMA concentrations, different induction times and different cell seeding densities, and the induction effect using the above optimized protocols was confirmed by optical microscope, flow cytometry and Phorbol-Myristil-Acetate (PMA) was the other macrophage differentiation stimulus used. Dec 22, 2022 · This differentiation protocol has been previously reported to be the minimum amount of PMA sufficient to induce stable differentiation without undesirable gene upregulation upon secondary stimuli (20). 1A and B). However, there are several concentrations of PMA commonly used to drive the differentiation of monocytic cell line to macrophage. Subculturing THP-1 cells: This document comprises a subculturing protocol for THP-1 cells. THP-1 cells can be differentiated to macrophages using phorbol-12-myristate-13-acetate (PMA). Once differentiated in the presence of PMA, they can be polarized into M1 or M2 macrophages that express markers similarly to polarized macrophages obtained from freshly isolated monocytes. 1% 2-ME at 41,667 cells per mL. 1 B). Different PMA concentrations and incubation times were tested (data not shown). Add 10 uL of 100 ng/mL PMA (final concentration of 100 ng/mL), culture for 3-5 days. Jun 20, 2024 · Results: Clearly, PMA induced THP-1 differentiation occurs in adherent culture conditions more than nonadherent culture conditions by increasing CD11b expression up to 90%, which was not affected by PD98059 when cells were exposed to PMA first (post-PD) but inhibited when PD98059 was treated prior to PMA treatment (pre-PD). In vitro models of differing macrophage functions are useful since human monocyte-derived macrophages are short-lived, finite and vary from donor to donor. Despite the wide use of this model, differentiation protocols using phorbol 12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D 3 (1,25D 3) vary drastically between studies. Feb 11, 2021 · Induction of HL‑60 cells for five days produced the best results in terms of differentiation markers and cell viability. The effects of PMA on the activation of the Na (+)-dependent uridine transporter were inhibited by staurosporine, suggesting the involvement of protein kinase C. In this plot, FSC-A is indicative of cell size, while SSC-A reflects the intensity of cytoplasmic granularity Mar 14, 2012 · Background T lymphocytes are orchestrators of adaptive immunity. 18 Through an MTT cell proliferation assay, which can measure the reduction of tetrazolium into an insoluble formazan product in viable cells, 19 PMA-stimulated This application note demonstrates the rapid, easy, and efficient characterization of cell surface markers of the THP-1 cell differentiation model by using the BD Lyoplate screening panel on suspension THP-1 cells (monocytes) using flow cytometry and on PMA-differentiated THP-1 cells (macrophages) using high-content imaging. Here, we have described a basic protocol for Th17 cell generation. Sep 1, 2024 · The human monocytic THP-1 cell line is the most routinely employed in vitro model for studying monocyte-to-macrophage differentiation. However, the conditions used for differentiation THP-1 is a monocyte cell line isolated from the peripheral blood of an acute monocytic leukemia patient and can be used in immunology research. Phenotypic changes resulting from THP-1 differentiation are shown in Figure 2. Sep 9, 2022 · In order to evaluate the immunomodulatory potential of novel monofloral Irish honeys, THP-1 monocyte-derived macrophages provided a suitable cell-based model. Hi Ce Dou , would you mind telling me more details about the THP-1 differentiation protocol, like cell density and media used for osteoclasts differentiation? From my experience in RAW cells and other cells, cell density is important. For this, we explore neuronal differentiation following standard protocols using (i) RA and (ii) a combination of RA and PMA (RA/PMA). However, the conditions used for differentiation, particularly the concentration of PMA and the duration of treatment, vary widely. The medium was changed every 2 days. Furthermore, we established a polarization protocol yielding M1 and M2 cells induced by IFNγ / LPS or IL4 / IL13, respectively (Fig 1A and 1C). Firstly, we chose a protocol that uses PMA and Vit D 3, and a protocol that involves activation with PMA followed by stimulation with cytokines. Oct 1, 2020 · In order to further investigate the differentiation protocol used, the basic property of phagocytosis of PMA differentiated THP-1 cells (Kurynina et al. : HY-18739, MCE) to induce adherent differentiation, but how to do it specifically? I want to ask for a detailed protocol, including: Feb 4, 2020 · A modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophages differentiation for transformed cells. This protocol is using THP-1 cells, a Dec 1, 2022 · This indicates that PMA-induced macrophage differentiation progresses over the 48 h period of resting culture in PMA-free culture medium of the differentiation protocol for both monocytic cell lines. Naïve T cells may differentiate into Th1, Th2, Th17 or iTreg phenotypes, depending on environmental co-stimulatory signals. Jul 17, 2021 · Phorbol 12-Myristate 13-Acetate (PMA) Treatment Suppresses Cell Growth Accompanied by Cytotoxicity in THP-1-Differentiated Macrophages The cell fate of THP-1-differentiated macrophages shows heterogeneity of cell growth and apoptosis. Differential response to phorbol 12-myristate 13-acetate (PMA) by THP-1. In vitro generated Th1, Th2 and Th17 cells have helped us to understand the molecular mechanism of their differentiation and their effector functions. Download scientific diagram | Induction of THP-1 differentiation by PMA at different concentrations. Jul 17, 2023 · THP-1 monocyte, which can be differentiated into macrophages by PMA, is widely used in researches on pathogen infection and host innate immunity, but reports on the induction methods of PMA are different and lack a unified standard, and the transcriptome characteristics of macrophage compared with T … In this application note, we showcase the development of the ThawReady™ THP-1 NF-κB-Luc2 cell line and demonstrate that it consistently exhibits high post-thaw viability and maintains the desired functionality. The cells are easy to culture and THP-1 is a human monocytic cell line originally isolated from a 1-year old male subject with acute monocytic leukemia (Tsuchiya et al. The line is positive for myc oncogene expression. Also, the expr … Objective: The aim of this study was to characterize the phenotype of undifferentiated and differentiated SH-SY5Y cells using various differentiation protocols. We Following differentiation, cells were rested in PMA-free media and analyzed via flow cytometry for GFP expression (HIV reactivation) for 6 days post-differentiation, which marks one week after PMA The protocols I have read seem straightforward on differentiating HL-60 cells, however I am not having luck using 1. They are easy to obtain in high yields, can be stored by freezing, and. For most purpose, a pre-treatment with PMA (10-100 nM HL-60 cells spontaneously differentiate and differentiation can be stimulated by butyrate, hypoxanthine, phorbol myristic acid (PMA, TPA), dimethylsulfoxide (DMSO, 1% to 1. The result from stimulation of PMA and VitD3 differentiated cells with 10 μg/ml LPS is shown in Figure 1. Dec 1, 2017 · The differentiation protocol strongly affects the cell characteristics and response to stressors (Daigneault et al. THP-1 differentiation was carried out at a final concentration of 100 nM PMA (Sigma-Aldrich P1585-1MG) for 72 hr, followed by trypsinization and isolation of adherent THP-1 derived macrophages with TrypLE (Thermo Fisher). , 2023). This cell line is sensitive to culture conditions; e. Several monocyte-to-macrophage differentiation protocols exist, with phorbol 12-myristate-13-acetate (PMA) being the most commonly used and accepted method. May 1, 2021 · Abstract Introduction: THP-1 cells, a human leukemia monocytic cell line, differentiated by phorbol myristate acetate (PMA) are widely used as surrogate of human macrophages. Mar 31, 2022 · Macrophages differentiated from peripheral monocytes using standard CSF1 and CSF2 protocols for M2 and M1 precursors, respectively, were compared with THP-1-derived macrophages treated with PMA and with SC-derived macrophages differentiated either by CSF1, CSF2, or PMA according to different protocols. To date, the genetic control of CD14 upregulation during this process remains poorly studied. Monocytic differentiation can be induced with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)/ phorbol 12-m ristate 12-acetate [Sigma P 8139]. Jul 17, 2023 · THP-1 monocyte, which can be differentiated into macrophages by PMA, is widely used in researches on pathogen infection and host innate immunity, but reports on the induction methods of PMA are different and lack a unified standard, and the transcriptome characteristics of macrophage compared with THP-1 cells remains unclear. 8× 10 6 cells/mL in 12-well plates were incubated with PMA (100 ng/mL) for 48 h [42, 43]. Results In this study, we first established a protocol for infection of non-adherent monocyte-like THP-1 cells (i. Note: THP-1 cells should be maintained at a cell density between 5–8 x 105 cells/mL prior to cell differentiation. Currently there is no established model for anti-inflammatory THP-1 polarization. Apr 15, 2019 · I am working with the U937 cell line, which I wish to use for macrophage survival assays. Average cell volume changes during differentiation have been reported in a number of differentiating systems. During the cell differentiation, the reactive oxygen species (ROS) level May 12, 2025 · It is generally recommended to use PMA (Cat. May 25, 2022 · In addition, the protein expression levels of these cytokines were significantly higher in PMA-differentiated THP-1 cells than in THP-1 cells at all doses (Figure 1 (d)– (f)). Cells were lysed and lysates from both THP-1 PMA Differentiation Protocol and CD14 Cell Surface Marker Expression 3. 3% DMSO and/or 1uM ATRA (all trans retinoic acid). Cells THP-1 is a human monocytic cell line originally isolated from a 1-year old male subject with acute monocytic leukemia (Tsuchiya et al. Quantitative proteomic analysis of PMA-induced changes. After 20 h of culture with 200 nM PMA, the cells adhered to the dish bottom and had the morphological characteristics of macrophages (Fig. Jun 7, 2022 · Here, we relatively quantify the proteomes of cells grown under standard conditions and obtained from two differentiation protocols employing RA or a combination of RA and PMA. Sep 12, 2020 · Objectives: The differentiation of THP-1 monocytes into macrophages is mainly conducted at a phorbol 12-myristate 13-acetate (PMA) concentration of 10–400 ng/ml. There is a plethora of differentiation protocols reported in the literature, with variations in both PMA concentration and time of treatment. , trypan blue staining + hemacytometer). In U937 cells with NRF2 silencing, PMA-stimulated cell adherence was significantly facilitated when compared to control U937 cells. Here we 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. The observation of cell morphology and the expression of CD68 and SR-A were performed to confirm the differentiation induced by PMA. Mar 1, 2016 · The human monocytic cell line, THP-1, is the most widely used model for primary human monocytes/macrophages. Mar 1, 2016 · These data indicate that exposure of monocytic THP-1 cells to 25 nM PMA over 48 h, followed by a recovery period of 24 h in culture in the absence of PMA, was the optimal protocol for the differentiation of THP-1 cells. The activation of NLRP3 inflammasome in macrophages has been proven to play a crucial role in the development of cardiovascular diseases. The cells are easy to culture and Mar 14, 2018 · Abstract THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) are widely used as a model for function and biology of human macrophages. We analyzed mRNA expression in un-induced, hemin-induced and PMA-induced K5 … May 12, 2025 · It is generally recommended to use PMA (Cat. glax2 ti6x 0w sgtv4 mbijep nejm lef 2t qel ne4vvy