scholarly journals Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy

2007 ◽  
Vol 2 (9) ◽  
pp. 2295-2301 ◽  
Author(s):  
Partha Mukhopadhyay ◽  
Mohanraj Rajesh ◽  
György Haskó ◽  
Brian J Hawkins ◽  
Muniswamy Madesh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Simultaneous Detection ◽  
Superoxide Production ◽  
Live Cells ◽  
Mitochondrial Superoxide

scholarly journals Simple quantitative detection of mitochondrial superoxide production in live cells

2007 ◽  
Vol 358 (1) ◽  
pp. 203-208 ◽  
Author(s):  
Partha Mukhopadhyay ◽  
Mohanraj Rajesh ◽  
Kashiwaya Yoshihiro ◽  
György Haskó ◽  
Pál Pacher
Keyword(s):  
Superoxide Production ◽  
Live Cells ◽  
Quantitative Detection ◽  
Mitochondrial Superoxide

scholarly journals Simultaneous quantitative detection of mitochondrial superoxide production and apoptosis in live cells.

2008 ◽  
Vol 22 (S1) ◽  
Author(s):  
Partha Mukhopadhyay ◽  
Mohanraj Rajesh ◽  
Kashiwaya Yoshihiro ◽  
Brian J Hawkins ◽  
Muniswamy Madesh ◽  
...  
Keyword(s):  
Superoxide Production ◽  
Live Cells ◽  
Quantitative Detection ◽  
Mitochondrial Superoxide

scholarly journals Simultaneous detection of decidual Th1/Th2 and NK1/NK2 immunophenotyping in unknown recurrent miscarriage using 8-color flow cytometry with FSC/Vt extended strategy

2017 ◽  
Vol 37 (3) ◽  
Author(s):  
Peng Dong ◽  
Xi Wen ◽  
Jia Liu ◽  
Cui-Yan Yan ◽  
Jing Yuan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mononuclear Cells ◽  
Recurrent Miscarriage ◽  
Simultaneous Detection ◽  
Live Cells ◽  
Color Flow ◽  
Multiparametric Flow Cytometry ◽  
Significant Difference

Th1/Th2 imbalance is considered as a mechanism for recurrent miscarriage. The NK1/NK2 paradigm is hypothesised to play an important role in pregnancy. However, few results showed simultaneous changes of these subsets in vivo in decidual tissues. The present study aimed to detect the decidual mononuclear cells (dMo), and the Th1/Th2, and NK1/NK2 paradigm simultaneously using multiparametric flow cytometry (MFC) in unexplained recurrent miscarriages (URM). Mononuclear cells were isolated from the decidual tissues of URM cases and early pregnant women. The mononuclear cell percent was demonstrated by detecting the expression of CD3, CD4, CD8, CD56, and CD16 extracellular markers, interferon (IFN)-γ, and interleukin (IL)-4 intracellular markers in live cells using 8-color flow cytometry with forward scatter (FSC)/side scatter (SSC) and FSC/viability (Vt) initial gating strategies, and the ratios of Th1/Th2 and decidual NK1 (dNK1)/decidual NK2 (dNK2) cells were compared between the subject groups. Two initial gating strategies of the FSC/SSC or FSC/Vt, with central or extended gating scales, were adapted, and there was no main effect or interaction for the cell proportions, except for the type 1 and type 2 subsets in the FSC/Vt extended gating strategy. There was no significant difference of the proportions of the decidual T, dNK, NKT-like, Th, and Tc cells between the two groups. However, the Th1/Th2 and dNK1/dNK2 ratios in the URM patients were higher compared with the normal group when using the FSC/Vt extended gating strategy. The present study provides means to detect Th1/Th2 and dNK1/dNK2 simultaneously in URM patients for large sample investigations in the future.

Abstract 599: Lighting Up P2Y12 Oligomers: Insights into the Role of Oligomerization in P2Y12 Function

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Aasma Khan ◽  
Jeffrey L Caplan ◽  
Donna Woulfe
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
G Protein ◽  
Time Lapse ◽  
Bimolecular Fluorescence Complementation ◽  
P2y12 Receptor ◽  
Live Cells ◽  
Bleeding Disorders ◽  
Mutant Forms

Introduction: Little is known about the role of P2Y12 oligomerization in receptor function and whether P2Y12 receptor mutations associated with human bleeding disorders may be explained by alterations in oligomerization. Objectives: 1) To determine whether P2Y12 homo- and hetero-oligomers are constitutive or dynamically regulated. 2) To explore whether P2Y12 mutants R256Q and R265W (previously detected in patients with abnormal bleeding, but with unaltered ADP binding) have different oligomerization affinities or kinetics and determine whether differences in P2Y12 oligomerization explain the functional defects. Methods: We employed a Venus-based Bimolecular Fluorescence Complementation (BiFC) approach in HEK293T cells transiently co-expressing P2Y12 or its mutant forms (R256Q or R265W) tagged with either the N-terminal (P2Y12-VN) or C terminal fragment (P2Y12-VC) of Venus, to characterize their homomeric interactions, in live cells using confocal microscopy and quantitative flow cytometry assays. Results: Agonist-independent formation of P2Y12 receptor homo-oligomers were detected on cell membranes. Time lapse imaging showed movement of P2Y12 receptor pairs from the endoplamic reticulum and Golgi network to the plasma membrane, suggesting that they are constitutive and required for export. Co-expression of P2Y12-VN with increasing amounts of P2Y12-VC demonstrated a dose-dependent increase in the fluorescence intensity of Venus, and reached saturation at a ratio of 1:3. Interestingly, the fluorescence intensities of homomeric P2Y12-R256Q-VN and R256Q-VC and, separately, P2Y12-R265W-VN and P2Y12R265W-VC were almost 4 times stronger than that of the wild type receptor as quantified in flow cytometry-based BiFC. Similar results were obtained in confocal microscopy. This suggests that these P2Y12 mutants form an increased number of dimers or oligomers with increased self-affinities. Conclusion: We demonstrate that P2Y12 forms constitutive homo-oligomers. Two mutations associated with bleeding disorders in patients have altered receptor-receptor interactions. Future investigation will explore the effect of mutations and receptor oligomers on G protein coupling and receptor: G protein stoichiometry.

scholarly journals Synthesis and evaluation of cell-permeable biotinylated PU-H71 derivatives as tumor Hsp90 probes

2013 ◽  
Vol 9 ◽  
pp. 544-556 ◽  
Author(s):  
Tony Taldone ◽  
Anna Rodina ◽  
Erica M DaGama Gomes ◽  
Matthew Riolo ◽  
Hardik J Patel ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Small Molecule ◽  
Cell Membranes ◽  
Systematic Approach ◽  
Affinity Purification ◽  
Hsp90 Inhibitor ◽  
Live Cells ◽  
Affinity Capture

The attachment of biotin to a small molecule provides a powerful tool in biology. Here, we present a systematic approach to identify biotinylated analogues of the Hsp90 inhibitor PU-H71 that are capable of permeating cell membranes so as to enable the investigation of Hsp90 complexes in live cells. The identified derivative 2g can isolate Hsp90 through affinity purification and, as we show, represents a unique and useful tool to probe tumor Hsp90 biology in live cells by affinity capture, flow cytometry and confocal microscopy. To our knowledge, 2g is the only reported biotinylated Hsp90 probe to have such combined characteristics.

scholarly journals Genomic instability induced by radiation-mimicking chemicals is not associated with persistent mitochondrial degeneration

2021 ◽  
Author(s):  
Luukkonen Jukka ◽  
Höytö Anne ◽  
Sokka Miiko ◽  
Syväoja Juhani ◽  
Juutilainen Jukka ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Ionizing Radiation ◽  
Genomic Instability ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Function ◽  
Mitochondrial Membrane ◽  
Neuroblastoma Cells ◽  
Superoxide Production ◽  
Mitochondrial Activity ◽  
Mitochondrial Superoxide

AbstractIonizing radiation has been shown to cause induced genomic instability (IGI), which is defined as a persistently increased rate of genomic damage in the progeny of the exposed cells. In this study, IGI was investigated by exposing human SH-SY5Y neuroblastoma cells to hydroxyurea and zeocin, two chemicals mimicking different DNA-damaging effects of ionizing radiation. The aim was to explore whether IGI was associated with persistent mitochondrial dysfunction. Changes to mitochondrial function were assessed by analyzing mitochondrial superoxide production, mitochondrial membrane potential, and mitochondrial activity. The formation of micronuclei was used to determine immediate genetic damage and IGI. Measurements were performed either immediately, 8 days, or 15 days following exposure. Both hydroxyurea and zeocin increased mitochondrial superoxide production and affected mitochondrial activity immediately after exposure, and mitochondrial membrane potential was affected by zeocin, but no persistent changes in mitochondrial function were observed. IGI became manifested 15 days after exposure in hydroxyurea-exposed cells. In conclusion, immediate responses in mitochondrial function did not cause persistent dysfunction of mitochondria, and this dysfunction was not required for IGI in human neuroblastoma cells.

scholarly journals KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

2021 ◽  
Author(s):  
Diana Spiegelberg ◽  
Jonas Stenberg ◽  
Pascale Richalet ◽  
Marc Vanhove
Keyword(s):  
Flow Cytometry ◽  
Live Cells ◽  
Time Resolved ◽  
Surface Receptors ◽  
Full Characterization ◽  
End Point ◽  
Titration Curves ◽  
Kinetics Of

AbstractDesign of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

scholarly journals Overexpression of Mitochondrial Leishmania major Ascorbate Peroxidase Enhances Tolerance to Oxidative Stress-Induced Programmed Cell Death and Protein Damage

2009 ◽  
Vol 8 (11) ◽  
pp. 1721-1731 ◽  
Author(s):  
Subhankar Dolai ◽  
Rajesh K. Yadav ◽  
Swati Pal ◽  
Subrata Adak
Keyword(s):  
Oxidative Stress ◽  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Ascorbate Peroxidase ◽  
Leishmania Major ◽  
Mitochondrial Respiratory Chain ◽  
Protein Carbonyl ◽  
Endonuclease G ◽  
Mitochondrial Membrane Depolarization ◽  
Induced Apoptosis

ABSTRACT Ascorbate peroxidase from Leishmania major (LmAPX) is one of the key enzymes for scavenging of reactive oxygen species generated from the mitochondrial respiratory chain. We have investigated whether mitochondrial LmAPX has any role in oxidative stress-induced apoptosis. The measurement of reduced glutathione (GSH) and protein carbonyl contents in cellular homogenates indicates that overexpression of LmAPX protects Leishmania cells against depletion of GSH and oxidative damage of proteins by H2O2 or camptothecin (CPT) treatment. Confocal microscopy and fluorescence spectroscopy data have revealed that the intracellular elevation of Ca2+ attained by the LmAPX-overexpressing cells was always below that attained in control cells. Flow cytometry assay data and confocal microscopy observation strongly suggest that LmAPX overexpression protects cells from H2O2-induced mitochondrial membrane depolarization as well as ATP decrease. Western blot data suggest that overexpression of LmAPX shields against H2O2- or CPT-induced cytochrome c and endonuclease G release from mitochondria and subsequently their accumulation in the cytoplasm. Caspase activity assay by flow cytometry shows a lower level of caspase-like protease activity in LmAPX-overexpressing cells under apoptotic stimuli. The data on phosphatidylserine exposed on the cell surface and DNA fragmentation results show that overexpression of LmAPX renders the Leishmania cells more resistant to apoptosis provoked by H2O2 or CPT treatment. Taken together, these results indicate that constitutive overexpression of LmAPX in the mitochondria of L. major prevents cells from the deleterious effects of oxidative stress, that is, mitochondrial dysfunction and cellular death.

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