Photoacoustic flow cytometry: principle and application for real-time detection of circulating single nanoparticles, pathogens, and contrast dyes in vivo

2007 ◽  
Vol 12 (5) ◽  
pp. 051503 ◽  
Author(s):  
Vladimir P. Zharov ◽  
Ekaterina I. Galanzha ◽  
Evgeny V. Shashkov ◽  
Jin-Woo Kim ◽  
Nikolai G. Khlebtsov ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Real Time ◽  
Real Time Detection

scholarly journals Real-time Detection of Circulating Apoptotic Cells by in Vivo Flow Cytometry

2005 ◽  
Vol 4 (4) ◽  
pp. 7290.2005.05148 ◽  
Author(s):  
Xunbin Wei ◽  
Dorothy A. Sipkins ◽  
Costas M. Pitsillides ◽  
John Novak ◽  
Irene Georgakoudi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Real Time ◽  
Apoptotic Cells ◽  
Real Time Detection ◽  
In Vivo Flow Cytometry

scholarly journals In vivo Raman flow cytometry for real-time detection of carbon nanotube kinetics in lymph, blood, and tissues

2009 ◽  
Vol 14 (2) ◽  
pp. 021006 ◽  
Author(s):  
Alexandru S. Biris ◽  
Ekaterina I. Galanzha ◽  
Zhongrui Li ◽  
Meena Mahmood ◽  
Yang Xu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Carbon Nanotube ◽  
Real Time ◽  
Real Time Detection

Real-time monitoring of circulating tumor cell release during tumor manipulation using in vivo photoacoustic and fluorescent flow cytometry

Head & Neck ◽  
2013 ◽  
Vol 36 (8) ◽  
pp. 1207-1215 ◽  
Author(s):  
Mazen A. Juratli ◽  
Mustafa Sarimollaoglu ◽  
Eric R. Siegel ◽  
Dmitry A. Nedosekin ◽  
Ekaterina I. Galanzha ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tumor Cell ◽  
Real Time ◽  
Circulating Tumor Cell ◽  
Real Time Monitoring ◽  
Cell Release

A reversible near-infrared fluorescence probe for the monitoring of HSO3–/H2O2 regulated cycles in vivo

2021 ◽  
Author(s):  
Qiuying Song ◽  
Bo Zhou ◽  
Dongyu Zhang ◽  
Haijun Chi ◽  
Hongmin Jia ◽  
...  
Keyword(s):  
Real Time ◽  
Near Infrared ◽  
Fluorescence Probe ◽  
Redox Homeostasis ◽  
Research Field ◽  
Fluorescence Probes ◽  
Detection Capability ◽  
Near Infrared Fluorescence ◽  
Real Time Detection

The development of well-designed fluorescence probes for the monitoring redox homeostasis in biosystems has become a desired research field owing to their noninvasive and real-time detection capability in vivo. In...

Long-Term Increase in Fetal Hemoglobin Expression in Nonhuman Primates Following Transplantation of Autologous Bcl11a Nuclease-Edited HSCs

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2035-2035 ◽  
Author(s):  
Olivier Humbert ◽  
Hans-Peter Kiem
Keyword(s):  
Flow Cytometry ◽  
Real Time ◽  
Real Time Pcr ◽  
Peripheral Blood ◽  
Nonhuman Primates ◽  
Pcr Analysis ◽  
Beta Globin ◽  
Gamma Globin ◽  
Cd34 Cells

Abstract Elevated levels of fetal hemoglobin (HbF) ameliorate the clinical symptoms of beta-thalassemia and sickle cell anemia. The transcription factor B-cell lymphoma/leukemia 11A (BCL11A) is required for silencing of gamma-globin expression in adult erythroid cells and functions as a switch from fetal to adult hemoglobin production in humans. BCL11A therefore constitutes a therapeutic target for the treatment of hemoglobinopathies. We inactivated BCL11A function by double-strand DNA break-induced mutagenesis using Transcription Activator-Like Effector Nucleases (TALENs). 20 to 30% gene editing could be achieved in vitro in human and nonhuman primate CD34+ cells by TALEN mRNAs electroporation targeting exon 2 of Bcl11a. Colony-forming efficiency was slightly lower in Bcl11a-edited CD34+ cells but lineage differentiation potential was unchanged. Erythroid differentiation of CD34+ cells in culture showed increased Fetal to Beta hemoglobin ratio in both human and primate Bcl11a-modified cells as compared to control cells, thus validating our editing approach to increase HbF production. To determine if Bcl11a-edited hematopoietic stem cells (HSCs) could be engrafted and give rise to HbF-producing erythrocytes, we transplanted a pigtail macaque with autologous CD34+ electroporated with Bcl11a TALEN mRNA following conditioning by total body irradiation. We detected about 1 % gene disruption in vivo early post-transplant and disruption frequency gradually declined to reach a set point of about 0.3% starting at day 28 post-transplantation. In this analysis, which we have so far taken out to 42 days, single clones could be tracked based on their mutation signature, and we found that several clones persisted over time, confirming engraftment of Bcl11a-modified cells. Since the transplantation procedure and chemo-radiotherapy conditioning can raise HbF production, three control animals that were transplanted using similar conditions as with the Bcl11a-edited HSCs and one untransplanted animal were also included in our analysis. Flow cytometry measurement of HbF in peripheral blood showed a rapid increase in F-cell production in all animals, reaching levels that ranged from 10% to 40% by 30 days, while the untransplanted control showed basal HbF expression of about 0.5% (Fig. 1A). The peak for HbF expression lasted for about 140 days and eventually returned to basal levels that averaged 0.5% for all control animals. In comparison, the animal transplanted with Bcl11a-edited cells showed significantly higher HbF levels starting at day 140 post-treatment (1-1.5%), and HbF production has remained constant for at least 150 days. This result was confirmed by hemoglobin mRNA analysis in peripheral blood using real-time PCR. We found a rapid increase in gamma globin expression following transplantation, before returning to near basal levels. As compared to controls, the animal transplanted with Bcl11a-edited cells showed a 5 to 10-fold increase in gamma to beta globin ratio at day 140 and this ratio has remained constant ever since (Fig. 1B). We are currently working on ways to enhance Bcl11a-editing and to select for Bcl11a-modified HSCs using targeted integration of the chemoselection cassette P140K MGMT to ultimately achieve curative HbF production. Potential TALEN off-target sites will also be examined as well as any side effect associated with the inactivation of BCL11A. Overall, our data demonstrate that transplantation of Bcl11a-edited HSCs results in elevated HbF production in nonhuman primates. Furthermore, we show that nonhuman primates can serve as a useful model for novel gene editing strategies toward the treatment of hemoglobinopathies. Figure 1. In vivo monitoring of HbF expression by flow cytometry and real-time PCR. (A) Intracellular HbF staining of peripheral blood measured by flow cytometry. (B) Real-time PCR analysis of hemoglobin transcripts in RNA isolated from peripheral blood. Expression was normalized to GAPDH and %HbG is calculated as HbG/(HbG+HbB). HbG=gamma globin; HbB=beta globin. Black line=Bcl11a transplant; grey line=control transplant; dashed line=untransplanted control. Figure 1. In vivo monitoring of HbF expression by flow cytometry and real-time PCR. (A) Intracellular HbF staining of peripheral blood measured by flow cytometry. (B) Real-time PCR analysis of hemoglobin transcripts in RNA isolated from peripheral blood. Expression was normalized to GAPDH and %HbG is calculated as HbG/(HbG+HbB). HbG=gamma globin; HbB=beta globin. Black line=Bcl11a transplant; grey line=control transplant; dashed line=untransplanted control. Disclosures No relevant conflicts of interest to declare.

scholarly journals Sublytic C5b-9 Induces Glomerular Mesangial Cell Apoptosis Through miR-3546/SOX4/Survivin Axis in Rat Thy-1 Nephritis

2018 ◽  
Vol 49 (5) ◽  
pp. 1898-1917 ◽  
Author(s):  
Chunyan Yao ◽  
Fengxia He ◽  
Longfei Liu ◽  
Zhiwei Zhang ◽  
Chenhui Zhao ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Real Time ◽  
Gene Transcription ◽  
Real Time Pcr ◽  
Mesangial Cell ◽  
Survivin Expression ◽  
Luciferase Reporter ◽  
Glomerular Mesangial Cell

Background/Aims: The activation of complement system and the formation of C5b-9 complex have been confirmed in the glomeruli of patients with mesangioproliferative glomerulonephritis (MsPGN). However, the role and mechanism of C5b-9-induced injury in glomerular mesangial cell (GMC) are poorly understood. Rat Thy-1N is an animal model for studying MsPGN. It has been revealed that the attack of C5b-9 to the GMC in rat Thy-1N is sublytic, and sublytic C5b-9 can cause GMC apoptosis, but the underlying mechanism is not fully elucidated. To explore the role and regulatory mechanism of C5b-9 in MsPGN lesion, we used rat Thy-1N model and first detected the change of microRNA (miRNA) profiles both in Thy-1N rat renal tissues (in vivo) and in the cultured GMCs with sublytic C5b-9 stimulation (in vitro). Then we determined the effect of miR-3546, which increased both in vivo and in vitro, on GMC apoptosis upon sublytic C5b-9 as well as the involved mechanism. Methods: Rat Thy-1N model was established and GMCs were treated with sublytic C5b-9. The rat renal cortex and the stimulated GMCs were obtained for miRNA microarray detection. Subsequently, the increased miRNAs were verified by real-time PCR. Meanwhile, to ascertain the ability of some miRNAs to upregulate cleaved caspase 3 and induce GMC apoptosis, the corresponding miRNA mimics were transfected into GMCs, followed by western blotting (WB) and flow cytometry mesurement. Thereafter, the miR-3546-targeted gene (SOX4) was predicted using bioinformatics approaches, and SOX4 expression in Thy-1N tissues and in the GMCs upon sublytic C5b-9 stimulation or miR-3546 mimic/inhibitor transfection were detected using real-time PCR and WB. To prove that miR-3546 can affect SOX4 gene transcription and SOX4 can regulate survivin expression, dual luciferase reporter assay, real-time PCR, WB and chromatin immunoprecipitation (ChIP) assays were performed. Furthermore, the role of miR-3546/SOX4/survivin axis in the GMC apoptosis induced by sublytic C5b-9 was examined using WB and flow cytometry. Results: Compared with normal renal tissues and untreated GMCs, there were 43 and 62 upregulated miRNAs (> 2-fold) in Thy-1N tissues and sublytic C5b-9-stimulated GMCs respectively. A total of 17 miRNAs were increased both in vivo and in vitro, 11 of which were validated by real-time PCR. Among them, miR-3546 could markedly promote GMC apoptosis and inhibit SOX4 or survivin expression in response to sublytic C5b-9, and either SOX4 or survivin overexpression markedly rescued the GMC apoptosis mediated by miR-3546 mimic. Additionally, SOX4 overexpression could reverse the survivin suppression by miR-3546 mimic, and SOX4 could bind to survivin promoter (-1,278 to -853 nt) and activate survivin gene transcription. Conclusion: MiR-3546/ SOX4/survivin axis has a promoting role in the GMC apoptosis triggered by sublytic C5b-9, and our findings may provide a new insight into the pathogenesis of rat Thy-1N and human MsPGN.

scholarly journals Shikonin Derivatives from Onsoma visianii Decrease Expression of Phosphorylated STAT3 in Leukemia Cells and Exert Antitumor Activity

Nutrients ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 1147
Author(s):  
Zeljko Todorovic ◽  
Jelena Milovanovic ◽  
Dragana Arsenijevic ◽  
Nenad Vukovic ◽  
Milena Vukic ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Death ◽  
Antitumor Activity ◽  
Real Time ◽  
Leukemia Cells ◽  
Antitumor Effects ◽  
Shikonin Derivatives ◽  
Phosphorylated Stat3

Antitumor effects of shikonins on chronic lymphocytic leukemia (CLL) and B-cell prolymphocytic leukemia (B-PLL) are mostly unexplored. The antitumor activity of shikonins, isolated from Onosma visianii Clem (Boraginaceae), in BCL1, mouse CLL cells and JVM-13, human B-PLL cells was explored in this study. The cytotoxicity of shikonin derivatives was measured by an MTT test. Cell death, proliferation, cell cycle, and expression of molecules that control these processes were analyzed by flow cytometry. Expression of STAT3-regulated genes was analyzed by real-time q-RT-PCR (Quantitative Real-Time Polymerase Chain Reaction). The antitumor effects of shikonin derivatives in vivo were analyzed, using flow cytometry, by detection of leukemia cells in the peripheral blood and spleens of mice intravenously injected with BCL1 cells. The two most potent derivatives, isobutyrylshikonin (IBS) and α-methylbutyrylshikonin (MBS), induced cell cycle disturbances and apoptosis, inhibited proliferation, and decreased expression of phospho-STAT3 and downstream-regulated molecules in BCL1 and JVM-13 cells. IBS and MBS decreased the percentage of leukemia cells in vivo. The link between the decrease in phosphorylated STAT3 by MBS and IBS and BCL1 cell death was confirmed by detection of enhanced cell death after addition of AG490, an inhibitor of Jak2 kinase. It seems that IBS and MBS, by decreasing STAT3 phosphorylation, trigger apoptosis, inhibit cell proliferation, and attenuate leukemia cell stemness.

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