Flow‐cytometric analysis of reactive oxygen species in peripheral blood mononuclear cells of patients with thyroid dysfunction

Sarkar, Mita; Varshney, Rajeev K.; Chopra, Madhu; Sekhri, Tarun; Adhikari, Jawahar Singh; Dwarakanath, Bilikere S.

doi:10.1002/cyto.b.20082

Cited by 47 publications

(31 citation statements)

References 25 publications

(29 reference statements)

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“…Thus the DCF fluorescence intensity of the cells is an index of intracellular levels of ROS. By quantifying fluorescence, the production of ROS could be quantified [12][13][14][15][16]. After anoxia-reoxygenation, the cells in 3 groups were washed and resuspended in PBS, and immediately detected on a Coulter Epics XL flow cytometer with the use of EXPO32 ADC software (Beckman Coulter, Inc., Miami, USA).…”

Section: Flow Cytometry Analysismentioning

confidence: 99%

Antisense Oligodeoxynucleotide Against Tissue Factor Inhibits Human Umbilical Vein Endothelial Cells Injury Induced by Anoxia-Reoxygenation

Yin

Luo²,

Yao³

et al. 2010

Cell Physiol Biochem

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Aims: The role of antisense oligodeoxynucleotide against tissue factor (aODN/TF) in cultured human umbilical vein endothelial cells (HUVECs) subjected to anoxia-reoxygenation (A/R) was investigated. Methods: HUVECs were divided randomly into control group, A/R group, aODN/TF+A/R group, sense oligodeoxynucleotide (sODN/TF) + A/R group and mismatched oligodeoxynucleotide (mODN/TF) + A/R group, in the latter 3 groups, HUVECs were transfected with aODN/TF, sODN/TF and mODN/TF respectively. HUVECs in all A/R groups underwent 3 hrs of anoxia and followed by 2 hrs of reoxygenation. In order to investigate the potential mechanisms of how increased TF may contribute to A/R injury in HUVECs, another set of HUVECs were incubated with human recombinant active site blocked factor VII (FVIIai) during A/R. Results: After A/R, TF expression at both mRNA and protein level was increased, furthermore, cell viability and the concentrations of SOD, GSH-PX and NO were declined, while LDH, MDA and ET-1 were overproduced (P<0.05 to 0.001 versus control group). In HUVECs of aODN/TF+A/R group, however, TF expression was inhibited, while the declined cell viability and the concentrations of SOD, GSH-PX, NO as well as the enhanced LDH, MDA and ET-1 levels occurred during A/R were ameliorated and reversed effectively (P<0.05 to 0.01 versus those in other A/R groups). The results also showed that ROS was increased and PAR-1, PAR-2, p38 MAP kinase and p42/44 MAP kinase were all activated after A/R (P<0.001 versus HUVECs under normoxia), while FVIIai inhibited the increment of ROS, PAR-1, PAR-2, p38 MAP kinase and p42/44 MAP kinase, and improved the changes of TF:C, MDA, SOD, GSH-PX, cell viability and LDH occurred during A/R (P<0.05 to 0.001 versus HUVECs without FVIIai treatment). Conclusion: Tissue factor plays an important role in the development of HUVECs injury induced by anoxia-reoxygenation, inhibition of TF with antisense oligodeoxynucleotide is an effective approach to ameliorate the damage.

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Section: Flow Cytometry Analysismentioning

confidence: 99%

Antisense Oligodeoxynucleotide Against Tissue Factor Inhibits Human Umbilical Vein Endothelial Cells Injury Induced by Anoxia-Reoxygenation

Yin

Luo²,

Yao³

et al. 2010

Cell Physiol Biochem

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“…Nevertheless, even if this technique is widely used, there is no agreement on the statistical analysis of cytometric data (17,18).…”

Section: Discussionmentioning

confidence: 99%

Modification to the Lampariello approach to evaluate reactive oxygen species production by flow cytometry

Brescia

Sarti

2007

Cytometry Pt A

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The aim of this article is to perform a statistical analysis of reactive oxygen species (ROS) cytometric data. It is demonstrated that the classical parametric and nonparametric statistical tests are not suitable to examine these data; the Kolmogorov-Smirnov test and the modification proposed by Lampariello are shown to be too sensitive with respect to the experimental bias (due to procedure or to the instrument) and variability in the ROS production within the repeated samples. Several approaches are examined and discussed. Modifications of the Lampariello's procedure are proposed to include the variability within samples. The validity of the proposed approach is verified by analyzing repeated measurements of ROS formation in cultured human lymphocytes untreated or treated with ferrous sulfate. The proposed approach is successful in considering the ''intersample'' variability in the ROS data analysis and keeps a good level of validity. Nevertheless, this procedure is not user-friendly and needs to be handled by an expert operator. ' 2007 International Society for Analytical Cytology Key terms flow cytometry; histogram comparison; Kolmogorov-Smirnov test; ROS production FLOW cytometry is a useful tool for the selective, fast, and accurate analysis of cells or particles (1), and although it is widely used in several application fields ranging from environmental monitoring (2) to cell biology, medicine, and toxicology, the comparison of cytometric data distributions remains an open question.In most cases, a single parameter is considered to characterize data sets. Untreated and treated samples are frequently compared by using the two-sample t test to asses the differences between two means. This test assumes that both samples came from normally distributed populations with equal variances (3), but this condition is often not fulfilled when flow cytometric (FC) data sets are considered.Evaluation of the results by the comparison of medians with a nonparametric test (i.e., paired Wilcoxon test) is free of the methodological inaccuracies, but provides insufficient information on the individual assays and their differences.In 1977, Young (4) introduced the nonparametric Kolmogorov-Smirnov (KS) statistical test (5) to FC data analysis, and now this test is commonly provided by most FC data-analysis programs.However, it is seldom used because the results are too sensitive to systematic shifts, making results statistically different when there is no biological difference.Lampariello (6) emphasized that an effective test should be able to identify the true differences resulting from the underlying biological phenomenon, while ignoring those caused by noise, sample-to-sample variability, and measurements effects. He proposed a modified KS test to evaluate cell ''positivity'' and to distinguish between statistical and biological differences.In this work, the FC frequency distributions of reactive oxygen species (ROS) production in human T leukemia cells (Jurkat) have been analyzed. Spontaneous and

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“…ROS production was detected using the dye 2', 7'-dichlorofluorescin diacetate (DCFH-DA; Sigma Aldrich) according to the procedure described previously (Sarkar et al, 2006). Briefly, cells were washed with PBS and then incubated with 10 µM DCFH-DA at 37°C for 30 min in the dark.…”

Section: Analysis Of Ros Levelsmentioning

confidence: 99%

Quercetin Iron (III) Complex Enhances Radiation-Induced Cell Death in Human Erythroleukemic Cell Lines by Increasing the Generation of Intracellular ROS

Kantapan¹,

Daowtak²,

Gateprayoon³

et al. 2017

American Journal of Applied Sciences

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Quercetin iron (III) complex (IronQ) is a paramagnetic agent which exhibits several biological effects. Despite extensive studies about quercetin metal complex, the role of complexes to enhance the effect of radiation still remains unexplored. To this end, we determined whether and how IronQ sensitizes cancer cells to Ionizing Radiation (IR). Doxorubicin resistant leukemic cells (K562/Adr) and their parental cell lines (K562) were used in this study. After treated with IronQ and radiation exposure (1-6 Gy), clonogenic survival assay, apoptosis, cell cycle distribution, acidic vesicular organelle staining and intracellular Reactive Oxygen Species (ROS) generation were evaluated. Combined treatment of IronQ with radiation significantly reduced the cell survival rate in both cell lines compared to radiation exposure alone or the control group. The mechanism underlying the cell growth rate inhibition of IronQ with IR was inducing cell arrest at G2/M phase. Moreover, IronQ pretreatment enhanced radiation-induced apoptosis in both cell lines with a greater efficacy in sensitive cell. Interestingly, increased intracellular ROS and accumulation of Acridine Orange (AO) in acidic organelle compartments in response to IronQ treatment combined with IR were observed 24 h post-treatment. In the context of our present findings demonstrating that IronQ in combination with radiation potentially improves the radiotherapeutic efficacy, the obtained results serve as a new strategy in the development of theranostic medicine.

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Flow‐cytometric analysis of reactive oxygen species in peripheral blood mononuclear cells of patients with thyroid dysfunction

Cited by 47 publications

References 25 publications

Antisense Oligodeoxynucleotide Against Tissue Factor Inhibits Human Umbilical Vein Endothelial Cells Injury Induced by Anoxia-Reoxygenation

Antisense Oligodeoxynucleotide Against Tissue Factor Inhibits Human Umbilical Vein Endothelial Cells Injury Induced by Anoxia-Reoxygenation

Modification to the Lampariello approach to evaluate reactive oxygen species production by flow cytometry

Quercetin Iron (III) Complex Enhances Radiation-Induced Cell Death in Human Erythroleukemic Cell Lines by Increasing the Generation of Intracellular ROS

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