Browsing by Subject "flow cytometry"

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  • Rotgers, E.; Cisneros-Montalvo, S.; Jahnukainen, K.; Sandholm, J.; Toppari, J.; Nurmio, M. (2015)
    Accurate analysis and quantification of different testicular cell populations are of central importance in studies of male reproductive biology. The traditional histomorphometric and immunohistochemical methods remain the gold standard in studying the complex dynamics of the testicular tissue. Through past years advances have been made in the application of flow cytometry for the rapid analysis of testicular cell populations. Detection of DNA content and of surface antigens and fluorescent reporters have been widely used to analyze and sort cells. Detection of intracellular antigens can broaden the possibilities of applying flow cytometry in studies of male reproduction. Here, we report a detailed protocol for the preparation of rat testicular tissue for detection of intracellular antigens by flow cytometry, and a pipeline for subsequent data analysis and troubleshooting. Rat testicular ontogenesis was chosen as the experimental model to validate the performance of the assay using vimentin and gamma H2AX as intracellular markers for the somatic and spermatogenic cells, respectively. The results show that the assay is reproducible and recapitulates the rat testis ontogenesis.
  • Eronen-Rasimus, Eeva Liisa; Kaartokallio, Hermanni; Lyra, Christina; Autio, Riitta; Kuosa, Harri; Dieckmann, Gerhard S.; Thomas, David N. (2014)
  • Turunen, Antti; Kuuliala, Antti; Mustonen, Harri; Puolakkainen, Pauli; Kylänpää, Leena; Kuuliala, Krista (2021)
    Objectives Clinical practice lacks biomarkers to predict the severity of acute pancreatitis (AP). We studied if intracellular signaling of circulating leukocytes could predict persistent organ dysfunction (OD) and secondary infections in AP. Methods A venous blood sample was taken from 174 patients with AP 72 hours or less from onset of symptoms and 31 healthy controls. Phosphorylation levels (p) of appropriately stimulated signal transducer and activator of transcription 1 (STAT1), STAT6, nuclear factor-kappa B (NF-kappa B), Akt, and nonstimulated STAT3 in monocytes, neutrophils, and lymphocytes was measured using phosphospecific flow cytometry. Results The patients showed higher pSTAT3 and lower pSTAT1, pSTAT6, pNF-kappa B, and pAkt than healthy controls. pSTAT3 in all leukocyte subtypes studied increased, and pSTAT1 in monocytes and T cells decreased in an AP severity-wise manner. In patients without OD at sampling, high pSTAT3 in monocytes and T lymphocytes were associated with development of persistent OD. In patients with OD, low interleukin-4-stimulated pSTAT6 in monocytes and neutrophils and Escherichia coli-stimulated pNF-kappa B in neutrophils predicted OD persistence. High pSTAT3 in monocytes, CD8(+) T cells, and neutrophils; low pSTAT1 in monocytes and T cells; and low pNF-kappa B in lymphocytes predicted secondary infections. Conclusions Leukocyte STAT3, STAT1, STAT6, and NF-kappa Beta phosphorylations are potential predictors of AP severity.
  • Lindelöf, Anna-Emilia (Helsingin yliopisto, 2019)
    Tiivistelmä - Referat - Abstract Background. Platelets are known to contain ample amounts of brain derived neurotrophic factor. Previous spectrophotometric studies carried out in Pia Siljander’s lab have shown that BDNF is secreted from activated platelets packed in extracellular vesicles. For this project we wanted to 1) confirm that BDNF really is secreted in extracellular vesicles (EVs)2) find out how the choice of agonist affected the BDNF cargo of the platelet derived EVs, and 3) find out if the BDNF is packed into EVs of certain densities rather than others. Methods. The platelets were isolated from platelet concentrates by size exclusion chromatography. The isolated platelets were then activated by thrombin and collagen co-stimulation (TC) and by Ca2+ionophore, respectively. The platelet activation produced extracellular vesicles (PEVs) which were isolated by differential ultracentrifugation. The isolated PEVs were then analysed by flow cytometry, ELISA and Western blot for EV typical membrane surface proteins and for their BDNF content. As we were interested finding out whether BDNF is enriched in PEVs to certain populations, density gradient centrifugation was performed. These samples were also analysed by Western blot and by ELISA. The size distribution and concentration of PEVs in all samples was analysed by Nanoparticle tracking analysis. Results and conclusions. This study confirmed that platelets secrete PEVs as a response to agonists. PEVs with higher BDNF concentration were produced using TC co-stimulation as compared to PEVs derived from the Ca2+ionophore. The result implies that BDNF is actively packed into PEVs for instance as a thrombogenic response. Based on the density gradient results it seems that BDNF was packed into certain population of PEVs with a density between 1.112 g ml-1 and 1.132 g ml-1 corresponding to a particle diameter of less than 500 nm. The finding that BDNF is actively packed into TC co-stimulation derived PEVs of a certain population is interesting from a theragnostic point of view, since EVs are likely to be key players in the development of new cell-based therapies. Had there been more time, it would have been interesting to optimize both the density gradient protocol and the ELISA analysis. This optimization of methods would make the process more efficient, less prone to sample loss, not to mention that there would be less intra-assay variation.
  • Puutio, Johanna (Helsingin yliopisto, 2020)
    Extracellular vesicles (EVs) are phospholipid bilayer-enclosed nanoparticles that are secreted by eukaryotic and prokaryotic cells. EVs carry macromolecules and signalling molecules to adjacent cells and play an important role in intercellular communication under both pathologic and homeostatic conditions. Therefore, they have become of significant interest for their therapeutic, diagnostic and prognostic potential. EVs are small and highly heterogeneous in size, shape, cargo and membrane composition, posing several challenges for establishing analytical and clinical guidelines. Therefore, EV research requires standardized and robust methods for their separation and characterization. In this study physical and immunochemical methods were employed to characterize human platelet-derived EVs (pEVs) generated from platelets activated with different external biochemical stimuli. The platelet-activating effect of the pro-inflammatory S100A8/A9 protein complex and a combination of thrombin and collagen were studied with nano flow cytometry. The size distribution of pEVs was studied with nanoparticle tracking analysis (NTA) and asymmetrical flow field-flow fractionation (AF4), which represents a newly emerging method on the EV field. Finally, fluorescent labelling and co-localization analysis were employed to characterize membrane marker composition of pEVs and assess its usefulness as an analytic tool for EV research. We succeeded in providing new hints towards meaningful discoveries in platelet biology by characterizing the way platelets respond to inflammatory and hemostatic signals by shedding pEVs. When platelet activation markers are characterized with flow cytometry, the S100A8/A9 protein appeared to cause a shift in membrane activation markers when compared to the thrombin- collagen mix and the baseline control. Increased TLT-1 translocation and decreased integrin αIIbβ3 expression on pEV surfaces suggests that S100A8/A9 induced pEV secretion through differently packed platelet α-granules, rather than from the plasma membrane. An increase in TLT-1 expression compared to decreased P-selectin and αIIbβ3 suggests that S100A8/A9 stimulation shifts platelet phenotype towards secretion rather than aggregation. A protocol for small pEV separation with AF4-MALS was set up. With this method, subtle differences between small pEV populations were seen that were not distinguishable with NTA or flow cytometry. When investigated with AF4-MALS, S100A8/A9 induced pEVs appeared larger than those produced with thrombin- collagen activation. The mean particle sizes of the pEV populations obtained from activated platelets were generally also larger than those produced without an activator. We tested novel methods to detect subtle differences in small EV population sizes that are easily missed with conventional methods due to their technical limitations. A well-optimised AF4 protocol can detect different pEV subpopulations and is a promising tool for EV. In the future, when AF4 is combined with a MALS detector and a fraction collector, nanoimaging of fluorescently labelled EVs could be combined with it as a downstream application to obtain information on their versatile biological functions.
  • Jokinen, Viljami; Sidorova, Yulia; Viisanen, Hanna; Suleymanova, Ilida; Tiilikainen, Henna; Li, Zhilin; Lilius, Tuomas O.; Matlik, Kert; Anttila, Jenni E.; Airavaara, Mikko; Tian, Li; Rauhala, Pekka V.; Kalso, Eija A. (2018)
    Development of tolerance is a well known pharmacological characteristic of opioids and a major clinical problem. In addition to the known neuronal mechanisms of opioid tolerance, activation of glia has emerged as a potentially significant new mechanism. We studied activation of microglia and astrocytes in morphine tolerance and opioid-induced hyperalgesia in rats using immunohistochemistry, flow cytometry and RNA sequencing in spinal-and supraspinal regions. Chronic morphine treatment that induced tolerance and hyperalgesia also increased immunoreactivity of spinal microglia in the dorsal and ventral horns. Flow cytometry demonstrated that morphine treatment increased the proportion of M2-polarized spinal microglia, but failed to impact the number or the proportion of M1-polarized microglia. In the transcriptome of microglial cells isolated from the spinal cord (SC), morphine treatment increased transcripts related to cell activation and defense response. In the studied brain regions, no activation of microglia or astrocytes was detected by immunohistochemistry, except for a decrease in the number of microglial cells in the substantia nigra. In flow cytometry, morphine caused a decrease in the number of microglial cells in the medulla, but otherwise no change was detected for the count or the proportion of M1-and M2-polarized microglia in the medulla or sensory cortex. No evidence for the activation of glia in the brain was seen. Our results suggest that glial activation associated with opioid tolerance and opioid-induced hyperalgesia occurs mainly at the spinal level. The transcriptome data suggest that the microglial activation pattern after chronic morphine treatment has similarities with that of neuropathic pain. (C) 2018 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Mannerström, Bettina; Kornilov, Roman; Abu-Shahba, Ahmed G.; Chowdhury, Iftekhar M.; Sinha, Snehadri; Seppänen-Kaijansinkko, Riitta; Kaur, Sippy (2019)
    Extracellular vesicles (EVs) are central to intercellular communication and play an important role in cancer progression and development. Osteosarcoma (OS) is an aggressive bone tumour, characterized by the presence of malignant mesenchymal cells. The specific tumour-driving genetic alterations that are associated with OS development are currently poorly understood. Mesenchymal stem cells (MSCs) of osteogenic lineage have been postulated as likely candidates as the cells of origin for OS, thus indicating that MSCs and OS stroma cells may be related cell types. Therefore, this study set out to examine the EV-mediated intercellular crosstalk of MSCs and OS. MSCs and pre-osteoblasts were treated with OS-EVs at different time points, and the epigenetic signature of OS-EVs was assessed by methylation analysis of LINE-1 (long interspersed element) and tumour suppressor genes. In addition, surface markers and expression of specific genes were also evaluated. Our data indicated that OS-EVs mediated LINE-1 hypomethylation in MSCs, whereas an opposite effect was seen in pre-osteoblasts, indicating that MSCs but not pre-osteoblasts were susceptible to epigenetic transformation. Thus, OS-EVs modulated the fate of MSCs by modulating the epigenetic status, and also influenced the expression of genes related to bone microenvironment remodelling. Overall, this study provided evidence that epigenetic regulation appears to be an early event in the transformation of MSCs during the development of OS. Elucidating the mechanisms of EV-mediated communication may lead to new avenues for therapeutic exploitation.
  • Nagy, Szabolcs; Polgar, Peter J.; Andersson, Magnus; Kovacs, Andras (2016)
    The aim of the present study was to test the FXCycle PI/RNase kit for routine DNA analyses in order to detect breeding bulls and/or insemination doses carrying cytogenetic aberrations. In a series of experiments we first established basic DNA histogram parameters of cytogenetically healthy breeding bulls by measuring the intraspecific genome size variation of three animals, then we compared the histogram profiles of bulls carrying cytogenetic defects to the baseline values. With the exception of one case the test was able to identify bulls with cytogenetic defects. Therefore, we conclude that the assay could be incorporated into the laboratory routine where flow cytometry is applied for semen quality control.
  • Sanmark, Enni; Wiksten, Johanna; Välimaa, Hannamari; Blomgren, Karin (2019)
    Aim: The purpose of this prospective study was to determine if there is a difference in number and distribution of salivary bacteria between patients with tonsillar infection and healthy volunteers. Background: The etiology of peritonsillar abscess (PTA) is unclear. Smoking, periodontal disease, and infection of minor salivary glands have been suggested as predisposing factors for PTA. Material and methods: Patients with acute tonsillitis (AT) (n = 54), peritonsillitis (PT) (n = 36), PTA (n = 58), and healthy volunteers (n = 52) were prospectively recruited and evaluated. Saliva bacteria were analyzed with flow cytometry. Patients and their treating physicians completed a questionnaire about patients' current disease, smoking habits, alcohol consumption, and oral health. Results: There were no differences in the total number of saliva bacteria between patients with acute throat infection and healthy volunteers (p = .104) or between AT, PT, and PTA patients (p = .273). Smoking habits, alcohol consumption, oral hygiene, or prior antibiotics had no effect on total amount of salivary bacteria in patients with acute throat infection. Conclusions: The effects of smoking on salivary bacteria do not seem to be the mechanism that promotes development of PTA in smokers.
  • Turunen, Antti; Kuuliala, Antti; Penttilä, Anne; Kaukonen, Kirsi-Maija; Mustonen, Harri; Pettilä, Ville; Puolakkainen, Pauli; Kylänpää, Leena; Kuuliala, Krista (2020)
    Activation of intracellular signaling pathways in circulating leukocytes represents an early step in systemic immune-inflammatory response occurring e.g. in acute pancreatitis (AP) and sepsis. Previously, we found aberrations in the phosphorylation of leukocyte signaling proteins in patients with sepsis or AP (measured
  • Al-Rashed, F.; Ahmad, Z.; Iskandar, M.A.; Tuomilehto, J.; Al-Mulla, F.; Ahmad, R. (2019)
    Background/Aims: TNF-α-mediated pro-inflammatory phenotypic change in monocytes is known to be implicated in the pathogenesis of metabolic inflammation and insulin resistance. However, the mechanism by which TNF-α-induces inflammatory phenotypic shift in monocytes is poorly understood. Since long-chain acyl-CoA synthetase 1 (ACSL1) is associated with inflammatory monocytes/macrophages, we investigated the role of ACSL1 in the TNF-α-driven inflammatory phenotypic shift in the monocytes. Methods: Monocytes (Human monocytic THP-1 cells) were stimulated with TNF-α. Inflammatory phenotypic markers (CD16, CD11b, CD11c and HLA-DR) expression was determined with real time RT-PCR and flow cytometry. IL-1β and MCP-1 were determined by ELISA. Signaling pathways were identified by using ACSL1 inhibitor, ACSL1 siRNA and NF-κB reporter monocytic cells. Phosphorylation of NF-κB was analyzed by western blotting and flow cytometry. Results: Our data show that TNF-α induced significant increase in the expression of CD16, CD11b, CD11c and HLA-DR. Inhibition of ACSL1 activity in the cells with triacsin C significantly suppressed the expression of these inflammatory markers. Using ACSL-1 siRNA, we further demonstrate that TNF-α-induced inflammatory markers expression in monocytic cells requires ACSL1. In addition, IL-1b and MCP-1 production by TNF-α activated monocytic cells was significantly blocked by the inhibition of ACSL-1 activity. Interestingly, elevated NF-κB activity resulting from TNF-α stimulation was attenuated in ACSL1 deficient cells. Conclusion: Our findings provide an evidence that TNF-α-associated inflammatory polarization in monocytes is an ACSL1 dependent process, which indicates its central role in TNF-α-driven metabolic inflammation. © 2019 The Author(s).