Concentrations Of Phorbol 12-myristate 13-acetate Research Articles

B-075 Biophysical Changes of Leukocyte Activation (and NETosis) in the Cellular Host Response to Sepsis

Abstract Background During sepsis, neutrophils, upon activation, undergo biophysical and biochemical changes that may terminate in the release of neutrophil extracellular traps (NETs) (1–3). The IntelliSep test is a rapid sepsis diagnostic that assesses immune activation by quantifying biophysical properties of leukocytes from whole blood in <10 minutes. The test results in the IntelliSep Index (ISI), ranging between 0.1–10.0, stratified into three interpretation bands (Band 1, Band 2, and Band 3) based on the probability of the clinical syndrome of sepsis (4, 5). We investigated the correlation between the IntelliSep result and immunological changes observed during leukocyte activation through in-vitro studies and using clinical samples from non-septic and septic patients. Methods In-vitro Studies: Healthy blood samples were collected between April - August 2022 from 18 volunteers at a blood donor center. Aliquots of each blood sample were incubated for 10 minutes with phorbol myristate acetate (PMA) in phosphate-buffered saline at one of 3 concentrations: 0, 200, and 400 nM prior to the IntelliSep test (3 repeats per donor per concentration). Clinical Studies: Subjects with signs or symptoms of infection were enrolled from Emergency Departments (EDs) in three similar but distinct prospective cohort studies (February 2016—September 2019) (1, 2). The IntelliSep test was performed on a aliquot of fresh whole blood from each subject, and remnant plasma was prepared and frozen for later analysis. Plasma levels of neutrophil elastase-DNA (NE DNA) and citrullinated histone H3-DNA (Cit-H3 DNA) complexes were quantified using previously reported custom ELISAs 3,6. Subjects were retrospectively adjudicated as sick (adjudicated as septic with SOFA scores peaking on the day of enrollment compared to two subsequent days) and “healthy” (SOFA for day of enrollment <2, hospital length of stay <3 days). Results Significant increases in ISI scores were observed with increasing concentration of PMA in healthy blood samples (0 and 200: P < 10−10; 0 and 400: P < 10−10). The clinical analysis cohort consisted of 39 “sick” and 42 “healthy” subjects. Linear correlation was observed between the ISI and NE DNA and Cit-H3 DNA quantities with significant increases across ISI Interpretation Bands (Band 1 to Band 3: P < 0.001). Conclusion Correlation of increasing ISI and PMA concentration supports the hypothesis that the biophysical changes measured by the IntelliSep test result from leukocyte activation. Observed correlation between the ISI and NET quantities suggest that NET formation is one of the activation pathways that result in the biophysical changes measured in the ISI. Together these experiments support that biophysical changes of leukocyte activation measured using a cellular host response test could provide a window into a patient’s state of dysregulated immunity and may have the potential to aid ED physicians in timely diagnosis of sepsis.

Biophysical Changes of Leukocyte Activation (and NETosis) in the Cellular Host Response to Sepsis.

Sepsis, the leading cause of mortality in hospitals, currently lacks effective early diagnostics. A new cellular host response test, the IntelliSep test, may provide an indicator of the immune dysregulation characterizing sepsis. The objective of this study was to examine the correlation between the measurements performed using this test and biological markers and processes associated with sepsis. Phorbol myristate acetate (PMA), an agonist of neutrophils known to induce neutrophil extracellular trap (NET) formation, was added to whole blood of healthy volunteers at concentrations of 0, 200, and 400 nM and then evaluated using the IntelliSep test. Separately, plasma from a cohort of subjects was segregated into Control and Diseased populations and tested for levels of NET components (citrullinated histone (cit-H3) DNA and neutrophil elastase (NE) DNA) using customized ELISA assays and correlated with ISI scores from the same patient samples. Significant increases in IntelliSep Index (ISI) scores were observed with increasing concentrations of PMA in healthy blood (0 and 200: p < 10-10; 0 and 400: p < 10-10). Linear correlation was observed between the ISI and quantities of NE DNA and Cit-H3 DNA in patient samples. Together these experiments demonstrate that the IntelliSep test is associated with the biological processes of leukocyte activation and NETosis and may indicate changes consistent with sepsis.

PMA-Treatment of Human Monocytes Induces a M1 Phenotype in Adherent Macrophages

Background: Human monocyte lines are widely used in basic research as model of inflammation, mostly following adherence with phorbol 12-myristate 13-acetate (PMA). However, the SC line, of normal human monocytes is not well documented, unlike tumour-derived cell lines, such as THP-1. Aim: The purpose of this study was to determine the phenotype of adherent macrophages, induced after the treatment with PMA in three different concentrations, starting from the most widely reported concentration in the literature. Methods: Normal human monocytes SC (ATCC CRL-9855) were routinely maintained according to manufacturer’s instructions. Cells were treated with Phorbol 12-myristate 13-acetate (PMA Sigma Aldrich P1585), in concentrations of 200 ng/mL, 100 ng/mL, 25 ng/mL and adhesion was documented using an Evos phase-contrast inverted microscope. Cell behaviour was validated by real-time impedance readings. The adhered cells were treated with bacterial lipopolysaccharide (LPS) in concentrations of 50 ng/mL (mimicking chronic inflammation) and 1 μg/mL (mimicking acute inflammation). The supernatant was collected twice, after 4 hours, respectively after 18 hours of treatment with LPS. A screening of pro- and anti-inflammatory cytokines was performed using the multiplexing platform Luminex 200. ELISA tests were performed to validate the cytokines secretion: IL-6, IL-8, IL-10, IL-23 and TNF-ɑ, using a LEGEND MAX Human ELISA kit specific to each cytokine. Results: Cell adhesion was studied by time-lapse microscopy for 48 hrs. The lowest concentration of PMA which induced cell adherence was 25 ng/mL. Multiplex screening of cytokines showed a pro-inflammatory phenotype of macrophages stimulated with LPS. This finding was validated by ELISA tests for IL-6, IL-8, IL-23 and TNF-ɑ (as pro-inflammatory cytokine) and IL-10 (an anti-inflammatory molecule). For the first category, we noticed a time-dependent response, present in adherent macrophages, but not in circulating monocytes. Regarding the second category of cytokines, the secretion is present only for the adhered and LPS treated cells. It is also present in a time-dependent manner (a higher concentration can be noticed in the collected supernatant after 18 hours of treatment compared with the one collected after 4 hours of treatment). Conclusion: The macrophages obtained from normal human monocytes with PMA are M1 type, regardless of the concentration used for differentiation.

Macrophage CCL22 Secretion Under Hypoxic Conditions Promotes the Metastasis of Triple-Negative Breast Cancer

Objective: To explore the mechanism by which macrophages secrete CCL22 to promote the metastasis of triple-negative breast cancer under hypoxic conditions. Methods: 20 ng/mL mass concentration of phorbol 12-myristate 13-acetate (PMA) cell culture medium, 4?,6-diamidino-2-phenylindole (DAPI), dimethyl sulfoxide (DMSO), trypsin digestion solution, CCL18 Kit , Interleukin (IL)-10 Kit, CCL17 Kit, CCL22 Kit, TRIzolTM Reagent Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Kit, triple-negative breast cancer cells MDA-MB-231 and BT-549, as well as other reagents were used to culture triple-negative breast cancer (TNBC) cells MDA-MB-231 and BT-549 as well as human mononuclear cells THP-1, analyze and observe the metastasis of triple-negative breast cancer cells to the lungs, the secretion of CCL22, the migration of triple-negative breast cancer, and the situation of CCR4. Results: Compared with normal tumor-associated macrophage (TAM), hypoxic TAM further promoted the migration of tumor cells. The number of tumor metastases in the lungs, induced by hypoxic TAM, was significantly higher than that of normoxic TAM. Hypoxia can significantly stimulate the expression of CCL22. CCL22 can significantly promote the migration of MDA-MB-231 and BT-549 cells. The expressions of CCR3, CCR4, and CCR5 in tumor tissues were significantly increased compared with normal tissues, in which CCR4 showed the most significant increase. Conclusion: TAM cultured under hypoxia significantly enhanced the migration ability of TNBC cells and promoted the metastasis of cancer cells to the lungs in vivo. The hypoxic condition induced TAM to secrete CCL22; the expression of CCL22 receptor, CCR4, in breast cancer tissues was significantly higher than that in normal tissues.

Normal human monocytic cell line CRL9855 can be transformed into macrophages with very low concentrations of phorbol 12-myristate 13-acetate

"Monocytes are leukocytes that can differentiate into tissue macrophages and act in immune surveillance and tissue reconstruction. They are frequently used in vitro to study inflammatory response, lymphocyte activation, and phagocytosis. A widely used model is the in vitro differentiation of various monocytic cell lines including THP-1 and U937 using phorbol 12- myristate 13-acetate (PMA). These cell lines have various limitations because of their malignant background. Data available on utilizing normal human monocytes cell lines are scarce. Our study aims to identify the optimum concentration of PMA needed to maximize the number of adherent macrophages using the normal monocytic cell line CRL-9855SC (ATCC CRL-9855). Using impedance readings and time-lapse microscopy, we determined that the optimum PMA concentration is 25ng/mL for 48 hourshrs. Using these optimized conditions, we were able to induce the formation of adherent and proliferating macrophages, which can be further used for morphology and functional studies."

Phorbol-12-myristate 13-acetate inhibits Nephronectin gene expression via Protein kinase C alpha and c-Jun/c-Fos transcription factors

Nephronectin (Npnt) is an extracellular matrix protein and ligand of integrin α8β1 known to promote differentiation of osteoblasts. A search for factors that regulate Npnt gene expression in osteoblasts revealed that phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), had a strong effect to suppress that expression. Research was then conducted to elucidate the signaling pathway responsible for regulation of Npnt gene expression by PMA in osteoblasts. Treatment of MC3T3-E1 cells with PMA suppressed cell differentiation and Npnt gene expression. Effects were noted at a low concentration of PMA, and were time- and dose-dependent. Furthermore, treatment with the PKC signal inhibitor Gö6983 inhibited down-regulation of Npnt expression, while transfection with small interfering RNA (siRNA) of PKCα, c-Jun, and c-Fos suppressed that down-regulation. The present results suggest regulation of Npnt gene expression via the PKCα and c-Jun/c-Fos pathway.

Comparative Proteomic Analysis Reveals Varying Impact on Immune Responses in Phorbol 12-Myristate-13-Acetate-Mediated THP-1 Monocyte-to-Macrophage Differentiation.

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. Several monocyte-to-macrophage differentiation protocols exist, with phorbol 12-myristate-13-acetate (PMA) being the most commonly used and accepted method. However, the concentrations and duration of PMA treatment vary widely in the published literature and could affect the probed phenotype, however their effect on protein expression is not fully deciphered. 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. Employing an integrated network analysis, we show that variations in PMA concentration and duration of rest post-stimulation result in downstream differences in the protein expression and cellular signaling processes. We demonstrate that these differences result in altered inflammatory responses, including variation in the expression of cytokines upon stimulation with various Toll-like receptor (TLR) agonists. Together, these findings provide a valuable resource that significantly expands the knowledge of protein expression dynamics with one of the most common in vitro models for macrophages, which in turn has a profound impact on the immune as well as inflammatory responses being studied.

Exploring the maturation of a monocytic cell line using self-organizing maps of single-cell Raman spectra.

Phorbol myristate acetate (PMA)-differentiated THP-1 cells are routinely used in lieu of primary macrophages to study macrophage polarization during host-pathogen interactions and disease progression. The phenotypes of THP-1 macrophages are influenced by the level and duration of PMA stimulation and possibly also by the presence of adhesion factors. Here, we use self-organizing maps (SOMs) of single-cell Raman spectra to probe the effects of PMA stimulation conditions and adhesion factors on THP-1 cell differentiation. Raman spectra encoding for biochemical composition were acquired from individual cells on substrates coated with fibronectin or poly-l-lysine before and after stimulation with 20 or 200 nM PMA for two different time intervals. SOMs constructed from these spectra showed the extent of spectral dissimilarity between different chronological cell populations. For all conditions, the SOMs indicated that the spectra acquired from cells after three-day treatment had diverged from those of untreated cells. The SOMs also showed that the higher PMA concentration produced both fully and partially differentiated cells for both adhesion factors after three days, whereas the outcome of stimulation for three days with the lower PMA concentration depended on the adhesion factor. On poly-l-lysine, treatment with 20 nM PMA for three days induced an intermediate stage of differentiation, but the same treatment produced partially and fully differentiated cells when applied to THP-1 cells on fibronectin. These results are consistent with the modulation of the transition of THP-1 monocytes into macrophage-like cells by integrin-binding interactions. Furthermore, differences in culture and stimulation conditions may confound comparison of results from separate studies.

The effects of Phorbol 12-myristate 13-acetate concentration on the expression of miR-155 and miR-125b and their macrophage function-related genes in the U937 cell line.

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. However, there are several concentrations of PMA commonly used to drive the differentiation of monocytic cell line to macrophage. Also, the expression of microRNA-155 (miR-155) and miR-125b in PMA-treated human monocytic cell line has not yet been reported. The five usual concentrations of PMA for stimulating macrophage differentiation are 10, 25, 50, 100, and 200 nM. In this study we compared the expression levels of miR-155, miR-125b and their related genes involved in macrophage functions in U937-derived cells after treatment with those five concentrations. The morphological study results showed that the five concentrations of PMA could induce macrophage differentiation in a similar manner. Moreover, cell proliferation and viability were not significantly different among these five conditions excepted the lower cell viability at 200 nM of PMA treatment. The five concentrations of PMA could upregulate the expression of miR-155 and miR-125b and increase the phagocytic activity of U937-derived cells in dose-reversal manner. The upregulation of miR-155 was correlated with increased expression levels of TNFα and decreased expression levels of BACH1 and CEBPβ, while the reduction of IRF4 was correlated with increased expression levels of miR-125b. Our study found that PMA could stimulate macrophage differentiation in a broad range of concentrations, however, the lower concentration could upregulate the higher expression of both miR-155 and miR-125b, and that correlated with the phagocytic functional activity of U937-derived macrophages.

Protein kinase C-mediated insulin receptor phosphorylation in diabetic rat retina.

Diabetic retinopathy (DR) involves a proliferation of vascular endothelial cells and loss of pericytes. There is a link among the action of protein kinase C (PKC) and insulin signaling. Thus, we investigated the differences between these cells in insulin receptor (IR) phosphorylation in DR. Retinas were removed from streptozotocin-induced diabetic or healthy rats, and IR expression levels were compared by immunoblot and immunohistochemistry. In vitro assays also were performed in order to determine the expressions of phosphorylated IR in both cells cultured under 5.5 or 25mM glucose by immunoblot. Cell viability was determined in both cells cultured under different concentrations of phorbol myristate acetate (PMA), a PKC activator. To determine the involvement of the PI3 kinase pathway of IR, PMA with or without wortmannin-induced changes in Akt was also analyzed. Immunoreactivity to the IR was decreased in diabetic retina. High glucose (25mM) increased phosphorylated IR levels in endothelial cells but not in pericytes. PMA (1nM or higher) induced death of pericytes, while endothelial cells were increased. PMA increased phosphorylated Akt in endothelial cells and decreased in pericytes. Wortmannin suppressed the PMA-induced phosphorylation of Akt in endothelial cells. The different responses to 25mM glucose and PMA were observed between retinal endothelial cells and pericytes. Thus, IR phosphorylation is likely important for retinal vascular cells to survive in diabetic retina.

Optimization of Experimental Settings for the Assessment of Reactive Oxygen Species Production by Human Blood.

The purpose of an experimental design is to improve the productivity of experimentation. It is an efficient procedure for planning experiments, so the data obtained can be analyzed to yield a valid and objective conclusion. This approach has been used as an important tool in the optimization of different analytical approaches. A D-optimal experimental design was used here, for the first time, to optimize the experimental conditions for the detection of reactive oxygen species (ROS) produced by human blood from healthy donors, a biological matrix that better resembles the physiologic environment, following stimulation by a potent inflammatory mediator, phorbol-12-myristate-13-acetate (PMA). For that purpose, different fluorescent probes were used, as 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), 2-[6-(4′-amino)-phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF), and 10-acetyl-3,7-dihydroxyphenoxazine (amplex red). The variables tested were the human blood dilution, and the fluorescent probe and PMA concentrations. The experiments were evaluated using the Response Surface Methodology and the method was validated using specific compounds. This model allowed the search for optimal conditions for a set of responses simultaneously, enabling, from a small number of experiments, the evaluation of the interaction between the variables under study. Moreover, a cellular model was implemented and optimized to detect the production of ROS using a yet nonexplored matrix, which is human blood.

Phorbol 12-Myristate 13-Acetate-Induced Changes in Chicken Enterocytes.

Increased intestinal epithelial permeability has been linked to many enteric diseases because it allows easy access of microbial pathogens and toxins into the system. In poultry production, the restrictions in the use of antibiotic growth promoters have increased the chances of birds being susceptible to different enteric diseases. Thus, understanding the mechanisms which compromise intestinal function is pertinent. Based on our previous observation which showed the primary chicken enterocytes in culture undergoing dystrophic changes on treatment with phorbol myristate acetate (PMA), we surmised that this model, which appeared to mimic increased intestinal permeability, may help to understand the mechanisms of this problem. As genomic and proteomic changes are associated with many physiological and pathological problems, we were interested to find whether certain proteomic changes underlie the morphological alterations in the enterocytes induced by PMA. We exposed primary enterocyte cultures to a sub-lethal concentration of PMA, extracted the proteins, and analyzed by mass spectrometry for differentially regulated proteins. Our results showed that PMA affected several biological processes which negatively affected their energy metabolism, nuclear activities, and differentially regulated the levels of several stress proteins, chaperon, cytoskeletal, and signal transduction proteins that appear to be relevant in the cause of enterocyte dystrophy. Phorbol myristate acetate-affected signal transduction activities also raise the possibilities of their increased susceptibility to pathogens. The changes in enterocyte integrity can make intestine vulnerable to invasion by microbial pathogens and disrupt gut homeostasis.

Abstract A74: T cell activation standardization for therapeutic assay development

Abstract Introduction: Several cancer immunotherapy strategies rely on T-cell activation (1). While T-cell activation mechanisms are well established, there is a dearth of quantified standardization of these activation pathways. Standardization with quantified end-points [induced by known activators and pathways] allows the development of useful assays to evaluate emerging immunotherapies. Our initial work has focused on T-cell activation. Materials and Methods: Activation of the Jurkat T-cell line was induced by the combined action of ionomycin and PMA (phorbol 12-myristate 13-acetate) (2). Jurkat cells were cultured in 24 well plates in RPMI 1640 media (with 10% FBS and 1% Penicillin/Streptomycin) at a density of 106 cells/ml. We looked at a wide concentration range for both activators (500 - 1500 ng/ml for ionomycin and 10 - 100 ng/ml for PMA), in order to identify their most synergistic combination for T-cell activation as quantified by biomarker expression (i.e. IL-2 ELISA). The negative controls were 1% DMSO (used for solubilizing the activators), and PBS (phosphate buffered saline). For a specific combination of 1000 ng/ml ionomycin and 100 ng/ml PMA, the designed time points were 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h. The cells and supernatant were preserved at the end of these time-points. The viability of the cells was tested using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay and the IL-2 production was detected using ELISA (Enzyme-Linked ImmunoSorbent Assay). Results: In addition to identifying the optimal activating concentrations of ionomycin and PMA, the obtained results characterized the time-course of maximal Jurkat cell activation over a 24 h period. During this 24 h period, the IL-2 levels and the cell proliferation rates progressively increased before starting to plateau. The production of IL-2 started increasing at 4 h from 157.6 pg/ml and continued to go up until 24 h to 687.6 pg/ml. Cell proliferation decreased a little during the first 2 h and increased from 57% to 114% (of the PBS control) between 8 to 24 h. Conclusions and Discussion: Our work establishes a standardized basis to determine the extent and the time-course of maximal T-cell activation through a given mechanism - in this case through the mechanism of calcium ionophore induced (ionomycin) and phorbol ester induced (PMA) T-cell activation (3). Such quantified standardization and maximization of T-cell activation in turn presents a cell-culture model (or assay) to evaluate candidate therapeutics designed to work through the same activation pathway. In addition to T-cells, we are working on the quantified standardization of the activation of NK-cells (4) and dendritic cells (5), with activation measured by the end-points of biomarker expression profiling (ELISA/Luminex), proliferation (MTT assay), microscopy and flow cytometry (CD receptor expression profiling).

Short communication: Recombinant bacteriophage endolysin PlyC is nontoxic and does not alter blood neutrophil oxidative response in lactating dairy cows

Mastitis is the leading cause of antimicrobial use on dairy farms. The potential for antimicrobial resistance has led to the examination of alternative strategies for controlling mastitis. One such alternative is PlyC, a potent peptidoglycan hydrolase derived from the streptococcal C1 bacteriophage that causes targeted lysis of the cell wall of Streptococcus uberis. At a concentration of 1.0 μg/mL, recombinant PlyC can induce lytic activity, suggesting that a low dose may successfully eliminate infection. We evaluated the dose effect of PlyC (1-50 µg/mL) on cytotoxicity and oxidative response on bovine blood polymorphonuclear leukocytes (PMN) obtained from 12 healthy, mid-lactation primiparous dairy cows. Following incubation at 0.5 and 2 h, cytotoxicity was characterized by measuring lactate dehydrogenase release from isolated cells. Oxidative burst response was characterized as the intensity of chemiluminescence produced in the interaction of reactive oxygen species generated in response to 0 or 1.6 µg/mL of phorbol 12-myristate-13-acetate (PMA) with a luminescent substrate with and without addition to PlyC to the incubation matrix. Data were analyzed as a complete randomized block design using mixed model procedures. Cytotoxicity of PlyC was not affected by concentrations up to 50 µg/mL. As expected, PlyC cytotoxicity on PMN varied across incubation time with greater cell toxicity measured at 2 h of incubation as compared with 0.5 h and is primarily attributed to the short life of PMN ex vivo. Concentrations of PlyC up to 50 µg/mL did not affect oxidative response; however, oxidative response was affected by incubation time and PMA concentration. In summary, varying doses of PlyC are nontoxic as estimated by lactate dehydrogenase release from cells and do not appear to alter PMA-stimulated reactive oxygen species production in bovine PMN. These early observations support continued work on the potential for application of this novel agent in combating mastitis.

The phorbol 12-myristate-13-acetate differentiation protocol is critical to the interaction of THP-1 macrophages with Salmonella Typhimurium.

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, the conditions used for differentiation, particularly the concentration of PMA and the duration of treatment, vary widely. Here we compare several differentiation conditions and compare the ability of THP-1 macrophages to interact with the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. The results show that THP-1 macrophages differentiated in high concentrations of PMA rapidly died following infection whereas those differentiated in low concentrations of PMA survived and were able to control the intracellular bacteria similar to primary human macrophages.

Sirt3 deficiency does not affect venous thrombosis or NETosis despite mild elevation of intracellular ROS in platelets and neutrophils in mice.

Inflammation is a common denominator in chronic diseases of aging. Yet, how inflammation fuels these diseases remains unknown. Neutrophils are the primary leukocytes involved in the early phase of innate immunity and inflammation. As part of their anti-microbial defense, neutrophils form extracellular traps (NETs) by releasing decondensed chromatin lined with cytotoxic proteins. NETs have been shown to induce tissue injury and thrombosis. Here, we demonstrated that Sirt3, a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase, an enzyme linked to human longevity, was expressed in mouse neutrophils and platelets. Using Sirt3-/- mice as a model of accelerated aging, we investigated the effects of Sirt3 deficiency on NETosis and platelet function, aiming to detect enhancement of thrombosis. More mitochondrial reactive oxygen species (ROS) were generated in neutrophils and platelets of Sirt3-/- mice compared to WT, when stimulated with a low concentration of phorbol 12-myristate 13-acetate (PMA) and a high concentration of thrombin, respectively. There were no differences in in vitro NETosis, with or without stimulation. Platelet aggregation was mildly augmented in Sirt3-/- mice compared to WT mice, when stimulated with a low concentration of collagen. The effect of Sirt3 deficiency on platelet and neutrophil activation in vivo was examined by the venous thrombosis model of inferior vena cava stenosis. Elevation of plasma DNA concentration was observed after stenosis in both genotypes, but no difference was shown between the two genotypes. The systemic response to thrombosis was enhanced in Sirt3-/- mice with significantly elevated neutrophil count and reduced platelet count. However, no differences were observed in incidence of thrombus formation, thrombus weight and thrombin-antithrombin complex generation between WT and Sirt3-/- mice. We conclude that Sirt3 does not considerably impact NET formation, platelet function, or venous thrombosis in healthy young mice.

Measurement of NET formation in vitro and in vivo by flow cytometry.

Neutrophil extracellular traps (NETs) are extracellular chromatin fibers adorned with antimicrobial proteins, such as myeloperoxidase (MPO), which are extruded from activated neutrophils. NETosis is the metamorphosis of neutrophils with NET formation that follows decondensation of DNA and rupture of the plasma membrane. Although NETs play important roles in innate immunity, excessive formation of NETs can be harmful to the hosts. Until now, various methods for evaluation of NETs have been reported. Although each has a virtue, the gold standard has not been established. Here we demonstrate a simple, objective, and quantitative method to detect NETs using flow cytometry. This method uses a plasma membrane‐impermeable DNA‐binding dye, SYTOX Green. SYTOX Green‐positive cells were detected in human peripheral polymorphonuclear cells exposed to a NET inducer, phorbol 12‐myristate 13‐acetate (PMA). The number of SYTOX Green‐positive cells was increased depending on the exposure duration and concentrations of PMA. Furthermore, co‐localization of MPO and plasma membrane‐appendant DNA of SYTOX Green‐positive cells was demonstrated. Moreover, a NET inhibitor, diphenylene iodonium, could significantly reduce the number of SYTOX Green‐positive cells induced by PMA. The collective evidence suggests that SYTOX Green‐positive cells include neutrophils that formed NETs. The established method could detect neutrophils that underwent NETosis but not early apoptosis with equivalence in quantification to another well‐used image analysis, which is based on fluorescent staining. Additionally, NETs that were formed in vivo were also detectable by this method. It is conceivable that the established method will bring us better understanding of the relation between NETosis and human diseases. © 2017 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus

The human monocytic cell line, THP-1, is the most widely used model for primary human monocytes/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. Despite the widespread use of THP-1 cells in studies elucidating macrophage responses to inflammatory stimuli, as well as the development and screening of potential therapeutics, there is currently no standardised protocol for the reliable differentiation of THP-1 monocytes to a macrophage phenotype using PMA. Consequently, reports using THP-1 cells have demonstrated significant phenotypic and functional differences between resultant THP-1 macrophage populations, which are largely attributable to the varying PMA differentiation methods used. Thus, to guarantee consistency and reproducibility between studies, and to ensure the relevance of THP-1 cells as an appropriate model for primary human macrophages, it is crucial to develop a standardised protocol for the differentiation of THP-1 macrophages. 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). Our results demonstrated that the function of the resultant THP-1 macrophage populations, as determined by tumour necrosis factor (TNF) secretion in response to LPS stimulation, varied significantly, and was dependent upon the concentration of PMA used to stimulate the differentiation of monocytes, and the period of rest following PMA exposure. These data indicate that exposure of monocytic THP-1 cells to 25nM PMA over 48h, 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.

Protein Kinase C Plays an Important Role in Exaggerated Vasoconstriction Associated with Insulin Deficiency but not Resistance

Deteriorated vascular reactivity has critical role in diabetic vascular complications. In the present study, the role of PKC on diabetes-induced deteriorated vascular reactivity was investigated. Insulin deficiency was induced by streptozotocin while its resistance by administering 10 % fructose. Isolated thoracic aorta reactivity to phenylephrine, KCl, acetylcholine and sodium nitroprusside was studied. The effects of in vitro incubation with the protein kinase C stimulant, phorbol 12-myristate 13-acetate or inhibitor, chelerythrine were also studied. Insulin deficiency increased responses to phenylephrine and KCl, decreased response to acetylcholine but showed no response to sodium nitroprusside. In vitro incubation with chelerythrine (7 µM, 20 min) normalized responses to KCl and phenylephrine but did not change response to acetylcholine or SNP. On the other hand, insulin resistance increased response to phenylephrine and KCl, impaired response to acetylcholine but did not affect the response to sodium nitroprusside, while in vitro incubation with chelerythrine did not change responses to phenylephrine, KCl, acetylcholine or sodium nitroprusside. In addition, protein kinase C stimulation by incubation of isolated normal aorta with different concentrations of phorbol 12-myristate 13-acetate (200, 400, 800 nM) for 1 h led to a similar impairment in isolated normal aorta to that in case of ID while co-incubation with chelerythrine restored normal vascular responses to KCl and phenylephrine. In conclusion, inhibition of protein kinase C by chelerythrine protects from the exaggerated vasoconstriction associated with insulin deficiency but not the resistance.

Identification of cell populations in splenocytes of Nile Tilapia (Oreochromis niloticus) and functional parameters of lymphocytes through flow cytometry.

Identification of cell populations in splenocytes of Nile Tilapia (Oreochromis niloticus) and functional parameters of lymphocytes through flow cytometry.