scholarly journals MicroRNA Quantitation During Dendritic Cell Endocytosis Using Imaging Flow Cytometry: Key Factors and Requirements

2018 ◽  
Vol 51 (2) ◽  
pp. 793-811
Author(s):  
Qiang Tong ◽  
Ying Zhu ◽  
Dandan Zhang ◽  
Qing Cai ◽  
Wenchun Qu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Key Factors ◽  
Argonaute 2 ◽  
Scanning Confocal Microscopy ◽  
Imaging Flow Cytometry ◽  
Microscopy Method ◽  
Stimulation Of

Background/Aims: MicroRNA (miRNA)-induced suppression of dendritic cells (DCs) has been implicated in many diseases. Therefore, accurate monitoring of miRNA endocytosis by DCs is important for understanding the role of miRNAs in many diseases. Recently, a method for measuring the co-localization of Argonaute 2 (AGO2)-associated miRNAs on laser-scanning confocal microscopy method was proposed to localize the miRNAs. But its definition was limited by the number of observed cells through its accuracy. Methods: In this study, a method based on imaging flow cytometry was developed to localize miR-590-5p with fluorescent probes in DCs. miR-590-5p proven to play an important role in tumor immunity. This method enabled the quantification, visualization and localization of the fluorescence intensity in 30,000 individual cells. Results: Using this method, the DCs with different endocytotic ability were distinguished. The behaviour of miR-590-5p during endocytosis under the stimulation of tumor antigen in DCs was observed, binding to its cognate target mRNA and degradation in DCs. Conclusion: This method based on imaging flow cytometry provide an additional method to study miRNA processing in DCs, which makes it a valuable addition to existing miRNA research techniques.

scholarly journals Peanut Clump Virus RNA-1-Encoded P15 Regulates Viral RNA Accumulation but Is Not Abundant at Viral RNA Replication Sites

2001 ◽  
Vol 75 (4) ◽  
pp. 1941-1948 ◽  
Author(s):  
Patrice Dunoyer ◽  
Etienne Herzog ◽  
Odile Hemmer ◽  
Christophe Ritzenthaler ◽  
Christiane Fritsch
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Laser Scanning Confocal Microscopy ◽  
Viral Rna ◽  
Epifluorescence Microscopy ◽  
Scanning Confocal Microscopy ◽  
Virus Rna ◽  
Green Fluorescent ◽  
Rna Accumulation

ABSTRACT RNA-1 of peanut clump pecluvirus (PCV) encodes N-terminally overlapping proteins which contain helicase-like (P131) and polymerase-like (P191) domains and is able to replicate in the absence of RNA-2 in protoplasts of tobacco BY-2 cells. RNA-1 also encodes P15, which is expressed via a subgenomic RNA. To investigate the role of P15, we analyzed RNA accumulation in tobacco BY-2 protoplasts inoculated with RNA-1 containing mutations in P15. For all the mutants, the amount of progeny RNA-1 produced was significantly lower than that obtained for wild-type RNA-1. If RNA-2 was included in the inoculum, the accumulation of both progeny RNAs was diminished, but near-normal yields of both could be recovered if the inoculum was supplemented with a small, chimeric viral replicon expressing P15, demonstrating that P15 has an effect on viral RNA accumulation. To further analyze the role of P15, transcripts were produced expressing P15 fused to enhanced green fluorescent protein (EGFP). Following inoculation to protoplasts, epifluorescence microscopy revealed that P15 accumulated as spots around the nucleus and in the cytoplasm. Intracellular sites of viral RNA synthesis were visualized by laser scanning confocal microscopy of infected protoplasts labeled with 5-bromouridine 5′-triphosphate (BrUTP). BrUTP labeling also occured in spots distributed within the cytoplasm and around the nucleus. However, the BrUTP-labeled RNA and EGFP/P15 very rarely colocalized, suggesting that P15 does not act primarily at sites of viral replication but intervenes indirectly to control viral accumulation levels.

scholarly journals Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein.

1996 ◽  
Vol 134 (2) ◽  
pp. 455-464 ◽  
Author(s):  
H J Matthies ◽  
H B McDonald ◽  
L S Goldstein ◽  
W E Theurkauf
Keyword(s):  
Laser Scanning ◽  
Spindle Assembly ◽  
Time Lapse ◽  
Laser Scanning Confocal Microscopy ◽  
Meiotic Spindle ◽  
Bipolar Spindle ◽  
Scanning Confocal Microscopy ◽  
Spindle Poles ◽  
Assembly Kinetics

We have used time-lapse laser scanning confocal microscopy to directly examine microtubule reorganization during meiotic spindle assembly in living Drosophila oocytes. These studies indicate that the bipolarity of the meiosis I spindle is not the result of a duplication and separation of centrosomal microtubule organizing centers (MTOCs). Instead, microtubules first associate with a tight chromatin mass, and then bundle to form a bipolar spindle that lacks asters. Analysis of mutant oocytes indicates that the Non-Claret Disjunctional (NCD) kinesin-like protein is required for normal spindle assembly kinetics and stabilization of the spindle during metaphase arrest. Immunolocalization analyses demonstrate that NCD is associated with spindle microtubules, and that the centrosomal components gamma-tubulin, CP-190, and CP-60 are not concentrated at the meiotic spindle poles. Based on these observations, we propose that microtubule bundling by the NCD kinesin-like protein promotes assembly of a stable bipolar spindle in the absence of typical MTOCs.

SINGLE CELL IMAGING OF BAX TRANSLOCATION DURING APOPTOSIS INDUCED BY PHOTOFRIN-PDT

2009 ◽  
Vol 02 (02) ◽  
pp. 209-214
Author(s):  
FEIFAN ZHOU ◽  
DA XING ◽  
WEI R. CHEN
Keyword(s):  
Cell Death ◽  
Mitochondrial Membrane ◽  
Laser Scanning ◽  
Cell Imaging ◽  
Laser Scanning Confocal Microscopy ◽  
Scanning Confocal Microscopy ◽  
Cellular Event ◽  
Single Cell Imaging ◽  
Induced Apoptosis

Apoptosis is an important cellular event that plays a key role in the therapy of many diseases. The mechanism of the initiation and regulation of photodynamic therapy (PDT)–induced apoptosis is complex. Our previous study found that Photofrin was localized primarily in mitochondria, the primary targets of Photofrin-PDT. The key role of Bax in the mitochondria-mediated apoptosis has been demonstrated in many systems. In order to determine the role of Bax in the mitochondrion-mediated apoptosis induced by Photofrin-PDT, we used the GFP-Bax plasmid to monitor the dynamics of Bax activation after PDT treatment. With laser scanning confocal microscopy, we found that Bax did not translocate from the cytosol to mitochondria when the mitochondrial membrane potential (ΔΨm) disappeared, measured by TMRM. Thus, for Photofrin-PDT, the commitment to cell death is independent of Bax activation.

Golgi apparatus is involved in intracellular Ca2+ regulation in epithelial LLC-PK1 cells

1995 ◽  
Vol 268 (5) ◽  
pp. C1133-C1140 ◽  
Author(s):  
X. Zha ◽  
S. Chandra ◽  
A. J. Ridsdale ◽  
G. H. Morrison
Keyword(s):  
Golgi Apparatus ◽  
Laser Scanning ◽  
Brefeldin A ◽  
Laser Scanning Confocal Microscopy ◽  
Hormonal Stimulation ◽  
Scanning Confocal Microscopy ◽  
Golgi Region ◽  
Calcium Green ◽  
Lines Of Evidence ◽  
Stimulation Of

Several lines of evidence suggest that the Golgi apparatus is involved in Ca2+ regulation in renal epithelial LLC-PK1 cells. Laser scanning confocal microscopy (LSCM) was employed to establish that a prominent perinuclear region is occupied mainly by the Golgi apparatus in this cell line. LSCM measurements in individual cells with the ionized Ca2+ indicator calcium green revealed that stimulation of LLC-PK1 cells with arginine vasopressin (AVP) resulted in the elevation of ionized Ca2+ levels. However, the vasopressin-induced rise in ionized Ca2+ was attenuated if the Golgi apparatus was disassembled by pretreating the cells with brefeldin A (BFA). Subcellular measurements of total Ca2+ with ion microscopy in cryogenically prepared cells indicated that 1) within 1 min of AVP treatment significant quantities of sequestered Ca2+ were released from the perinuclear Golgi region and 2) the BFA treatment reduced the total Ca2+ stored in the Golgi region. These observations indicate that the Golgi apparatus is sensitive to hormonal stimulation and may play important roles in intracellular Ca2+ regulation in LLC-PK1 cells.

Induction of Gamma Globin and Erythrocyte Anion Exchane Protein in BFU-E Cultured in the Presence of Hydroxyurea.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3755-3755
Author(s):  
Gloria A. Green ◽  
Beau R. Braden ◽  
Obianuju Mba ◽  
Stacy A. Chivira ◽  
Laleh Ramezani ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Band 3 ◽  
Band 3 Protein ◽  
Erythroid Progenitors ◽  
Gamma Globin ◽  
Scanning Confocal Microscopy ◽  
Reactive Mechanism

Abstract Hydroxyurea (HU) an S-phase specific cytotoxic agent has been used for the treatment of patients with sickle cell hemoglobinopathy and beta-thalassemias. The clinical efficacy of HU is due primarily to increases in fetal hemoglobin (HbF) levels. HU increases the %HbF and the %F cells. The HU reactive mechanism(s) in erythroid cells, however, have not been clearly defined. Patients receiving HU therapy develop subpopulations of macrocytic erythrocytes. Our previous studies demonstrate that sickle cell patients treated with HU develop subpopulations of RBCs that express greater relative levels of the erythrocyte anion exchange protein (AE1) per cell as compared with untreated individuals. We propose that part of the HU reactive mechanism will include the upmodulation of non-gamma globin erythroid proteins that contribute to the macrocytic structures. As part of our investigation of the development of RBCs expressing increased band 3 protein per cell, we have examined the possibility that HU induced AE1 synthesis can be detected in vitro using cultured erythroid progenitors. To investigate HU induced protein synthesis as a function of HU concentration, erythroid progenitors were cultured in semisolid media containing different concentrations of HU [0–40 micromolar] then assayed for AE1(band 3) and gamma globin. BFU-E were scored and harvested after 15 days in culture, then assayed. The change in the frequency of cells positive for band 3 protein was determined by flow cytometry, these cells were then assessed by laser scanning confocal microscopy. Results show that the frequency of cells positive for band 3 protein was greater in colonies grown in hydroxyurea as compared to controls. The band-3 upmodulation appears to plateau at 12.5 micromolar HU. These cells were assessed for dual and single stains by laser scanning confocal microscopy. Both band-3 protein and spectrin were detected. Laser scanning confocal microscopy revealed spectrin in the majority of cells; both band 3 and spectrin were detected in forty percent of the cells cultured in 12.5 micromolar HU. Band 3 protein detected by Western blots was increased [1–1.5 fold] over untreated control BFU-E harvested during the same time period. Secondly, the presence of band 3 protein and gamma globin in BFU-E was detected using two-color flow cytometry. BFU-E were cultured in increasing concentrations of HU. Colonies were permeabilized, and then labeled with tricolor-conjugated-anti-gamma globin. These cells were subsequently labeled with monoclonal anti-band 3 and PE-labeled anti-mouse antibody. Results show 2–3 fold increase in the % band 3 plus gamma globin positive cells over untreated cells. Collectively, these results suggest that part of the mechanism of HU action in erythroid cells involves the induction of erythroid structural proteins concordant with the induction of gamma globin.

Effects of [Ca2+]i, SR Ca2+ load, and rest on Ca2+ spark frequency in ventricular myocytes

1997 ◽  
Vol 272 (2) ◽  
pp. H657-H668 ◽  
Author(s):  
H. Satoh ◽  
L. A. Blatter ◽  
D. M. Bers
Keyword(s):  
Laser Scanning ◽  
Ventricular Myocytes ◽  
Gradual Increase ◽  
Rest Period ◽  
Laser Scanning Confocal Microscopy ◽  
Time Dependent ◽  
Contraction Coupling ◽  
Scanning Confocal Microscopy ◽  
State Fusion ◽  
Stimulation Of

In heart, spontaneous local increases in cytosolic Ca2+ concentration ([Ca2+]i) called "Ca2+ sparks" may be fundamental events underlying both excitation-contraction coupling and resting Ca2+ leak from the sarcoplasmic reticulum (SR). In this study, resting Ca2+ sparks were analyzed in rabbit and rat ventricular myocytes with laser scanning confocal microscopy and the fluorescent Ca2+ indicator fluo 3. During the first 20 s of rest after regular electrical stimulation, both the frequency of Ca2+ sparks and SR Ca2+ content gradually decreased in rabbit. When rabbit SR Ca2+ content was decreased by reduction of stimulation rate. the initial resting spark frequency was also decreased, even though resting [Ca2+]i was unchanged. The rest-dependent decrease in spark frequency in rabbit cells was prevented by inhibition of Na+/Ca2+ exchange (which also prevents SR Ca2+ depletion during rest). These results suggest that elevation of SR Ca2+ content can increase Ca2+ spark frequency. In contrast to rabbit cells, 20 s of rest produced a gradual increase in spark frequency in rat cells, although SR Ca2+ content was constant and Ca2+ influx was completely prevented. This indicates that there is a time-dependent increase in spark probability during rest that is independent of [Ca2+]i or SR Ca2+. This effect was also apparent in rabbit cells when SR Ca2+ depletion was prevented by blocking Na+/Ca2+ exchange. Stimulation of Ca2+ extrusion via Na+/Ca2+ exchange in the rat (by Ca2+-free superfusion, which slowly depletes SR Ca2+ content) converted the normal rest-dependent increase in spark frequency to a decrease. The amplitude of individual Ca2+ sparks increased with increasing SR Ca2+ content. In the Ca2+-overloaded state, fusion of sparks or long-lasting localized increases of [Ca2+]i were observed with increased spark frequency. We conclude that the resting frequency of Ca2+ sparks can be independently affected by changes in SR Ca2+ content, [Ca2+]i, or rest period. The latter may reflect recovery of the SR Ca2+ release channels from inactivation or adaptation.

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