scholarly journals A FRET-based fluorescent Zn2+ sensor: 3D ratiometric imaging, flow cytometric tracking and cisplatin-induced Zn2+ fluctuation monitoring

2020 ◽  
Vol 11 (40) ◽  
pp. 11037-11041
Author(s):  
Hongxia Xu ◽  
Chengcheng Zhu ◽  
Yuncong Chen ◽  
Yang Bai ◽  
Zhong Han ◽  
...  
Keyword(s):  
Real Time ◽  
Flow Cytometric ◽  
Ratiometric Imaging

A Zn2+ sensor exhibiting 3D ratiometric imaging and flow cytometric ability was constructed based on the FRET mechanism, and cisplatin-induced endogenous labile Zn2+ fluctuations were monitored in real time.

scholarly journals Real-Time Ratiometric Imaging of Micelles Assembly State in a Microfluidic Cancer-on-a-Chip

2020 ◽  
Author(s):  
Natalia Feiner-Gracia ◽  
Adrianna Glinkowska Mares ◽  
Marina Buzhor ◽  
Romen Rodriguez-Trujillo ◽  
Josep Samitier Marti ◽  
...  
Keyword(s):  
Real Time ◽  
Ratiometric Imaging

Ratiometric fluorescent probes for selective and sensitive visualization of bacterial microenvironment protease activity

2019 ◽  
Vol 55 (35) ◽  
pp. 5064-5067 ◽  
Author(s):  
Qinghua Wang ◽  
Xiang Wang ◽  
Ying Sun ◽  
Xiaoxiao Yang ◽  
Leilei Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Fluorescent Probes ◽  
Protease Activity ◽  
Ratiometric Imaging

Two novel FRET probes for selective real-time ratiometric imaging of SspA activity in live S. aureus cells.

scholarly journals Solubilization and Display of G Protein-Coupled Receptors on Beads for Real-Time Fluorescence and Flow Cytometric Analysis

BioTechniques ◽  
2000 ◽  
Vol 28 (5) ◽  
pp. 976-985 ◽  
Author(s):  
L.A. Sklar ◽  
J. Vilven ◽  
E. Lynam ◽  
D. Neldon ◽  
T.A. Bennett ◽  
...  
Keyword(s):  
Real Time ◽  
G Protein ◽  
Flow Cytometric Analysis ◽  
G Protein Coupled Receptors ◽  
Flow Cytometric ◽  
G Protein Coupled

Dynamic Assessment of Apoptosis for In Vitro Design of Bortezomib Combination Therapies for Lymphoid Malignancies.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2494-2494
Author(s):  
Lauren C. Wallis ◽  
Matthew J. Streetly ◽  
Rebecca Auer ◽  
John Gribben ◽  
Dean Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Real Time ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Growth Characteristics ◽  
Cytotoxic Agents ◽  
Alamar Blue ◽  
Apoptosis Induction ◽  
Flow Cytometric ◽  
Induction Of Apoptosis

Abstract Conventional techniques for assessing drug response and apoptosis induction rely on static assessment of cellular changes at predetermined time points (e.g. detection of exposed membrane phospholipids by Annexin V). The Kinetics of Optical Response assay (KOR) is a new technique that detects induction of apoptosis dynamically. It employs a spectrophotometric methodology to detect changes in optical density associated with membrane blebbing related to growth and death, allowing detection of apoptosis in real time. The KOR assay has already predicted the response to cytotoxic agents of AML cell lines and primary samples. This study uses the KOR assay in lymphoid malignancy and shows sensitivity to apoptosis induction by conventional and novel agents including bortezomib. The lymphoma cell line DOHH2 (t(14;18)), U266 (myeloma), K562 (CML) and primary CLL cells were used in this study with HL60 (AML) as a control. Cells were seeded in 96 well plates and treated with a variety of drugs alone or in combination (cytarabine, fludarabine, doxorubicin, daunorubicin, etoposide, melphalan, bortezomib) at multiple concentrations. Measurements were made at 5 min. intervals for up to 48 hrs and analysed using KORSoft™ software to generate apoptotic response curves. To validate this approach conventional techniques were used for comparison (Alamar Blue for cytotoxicity and flow cytometric analysis of cell cycle and apoptosis using propidium iodide and Annexin V staining respectively). The KOR assay can show changes in growth characteristics, induction of apoptosis and necrosis in response to drugs permitting a continuous analysis for maximum sensitivity (Smax). DOHH2 was found to be dose responsive to four of the drugs used, with the Smax for 10μM daunorubicin at 6 hours (48%), 1μM doxorubicin at 8 hours (38%), 100μM etoposide at 8 hours (52%), and minimally to 100μM cytarabine at 16 hours (21%). There was no effect from fludarabine. The addition of bortezomib increased Smax to 89% with etoposide and to a lesser degree with the other cytotoxic drugs. U266 showed a similar spectrum of results with greatest Smax with 100μM melphalan at 9 hours (57%) enhanced to 78% with the addition of bortezomib. There was minimal response to cytarabine and fludarabine. Parallel flow cytometric analysis using Annexin V and PI showed similar results to those from the KOR assay confirming the assessment of apoptosis to be valid. Cell cycle analysis showed an increased sub-G1 peak in keeping with apoptosis at times of Smax assessed by the KOR assay. The Alamar Blue cytotoxicity assay showed a dose dependent decrease in cell proliferation in response to increasing drug dose again paralleling other apoptosis measurements implying an apoptotic effect due to drug action and correlate well with those from the KOR assay. Primary CLL samples following CD19 selection were cultured with and without IL4 and exposed to the KOR assay with cytotoxics and bortezomib. Culture with IL4 alone gave good growth characteristics and revealed the combination of etoposide and bortezomib to provide the best induction of apoptosis (Smax 82%) compared to etoposide (26%) or bortezomib (32%) alone. The KOR assay is a microtitre approach to the assessment in real time of apoptosis. This study suggests the combination of bortezomib and etoposide is effective for lymphoma. Such approaches can accelerate the development of effective clinical trials.

The Inhibitory Receptor FcgRIIB Is Overexpressed, and Its Ligation by Anti- FcgRIIB Antibodies Suppresses IgM Production and Induces Apoptosis in Waldenstrom's Macroglobulinemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3736-3736 ◽  
Author(s):  
Guang Yang ◽  
Ping Gong ◽  
Lian Xu ◽  
Zachary Hunter ◽  
Jenny Sun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Real Time ◽  
Real Time Pcr ◽  
Flow Cytometric Analysis ◽  
Cross Linking ◽  
Inhibitory Receptor ◽  
Downstream Signaling ◽  
Flow Cytometric ◽  
Induction Of Apoptosis ◽  
Waldenström's Macroglobulinemia

Abstract Abstract 3736 Poster Board III-672 Introduction Waldenstrom's macroglobulinemia (WM) is a lymphoplasmacytic lymphoma characterized by overproduction of a monoclonal IgM paraprotein which can produce morbidity including hyperviscosity, as well as autoimmune related neuropathy, hemolysis, and thrombocytopenia. Therefore approaches aimed at both suppressing IgM production, as well as selectively inducing apoptosis of WM cells represent an ideal treatment strategy for WM. FcgRIIB is an inhibitory receptor that is expressed on B-cells, and whose expression we recently identified as highly over-expressed in WM. Importantly, FcgRIIB possesses an immunoreceptor tyrosine-based inhibitory motif (ITIM), and which becomes phosphorylated at Tyr 292 upon activation, and is then followed by inhibition of BCR signaling and induction of apoptosis. We therefore validated the expression of this receptor in WM, and examined the impact of its ligation on tumor cell killing, IgM secretion and downstream signaling events in WM cells. Patients and Methods Bone marrow lymphoplasmacytic cells (LPC) from 12 WM patients which were sorted for CD19+ and CD138+, and BCWM.1 WM cells were subjected to real-time PCR and flow cytometric analysis. Cells were then subjected to co-culture with anti- FcgRIIB (AT10, 7.3) or control antibodies for 24-48 hours, and their effects on survival, IgM production and downstream signaling were assessed. Results Real-time PCR and flow-cytometric analysis demonstrated strong expression of FcgRIIB in WM patient bone marrow CD19+ and CD138+ cells, thus confirming our recent microarray results. Importantly, the expression of FcgRIIB in WM LPC correlated with the memory B-cell marker CD27. Anti-FcgRIIB antibody treatment dramatically reduced constitutive, and/or IL-6 induced IgM production in CD19+ and CD138+ sorted primary WM LPC, as well as CD32hiCD138hi BCWM.1 cells. This effect was observed in some experiments at an early time point that had not effected survival. Among primary CD138+ WM LPC and CD32hiCD138hi expressing BCWM.1 cells, treatment with anti-FcgRIIB antibodies for 48 hours led to increased apoptosis in 10 of 12 patients, as assessed by Annexin V and PI staining which occurred despite blockade with a pan-caspase inhibitor, and was even more pronounced when anti-FcgRIIB antibodies were cross-linked. Western blot analysis revealed that treatment of CD32hiCD138hi expressing BCWM.1 cells with cross-linked anti- FcgRIIB antibodies led to phosphorylation of Tyr 292 of the FcgRIIB ITIM which was not observed in the absence of cross-linking. Binding of FcgRIIB by both the AT10 and 7.3 antibodies resulted in dephosphorylation of Akt, which was further reduced in the presence of cross-linking. Coincident with the above, the pro-apoptotic molecule JNK also underwent phosphorylation in the presence of anti-FcgRIIB binding. Conclusion Taken together, these studies validate our previous microarray data by showing that the inhibitory receptor FcgRIIB is strongly expressed on LPC from WM patients, and ligation thereof leads to suppression of IgM production and induction of apoptosis thereby identifying FcgRIIB as a novel therapeutic target in WM. Disclosures: No relevant conflicts of interest to declare.

Cancer cell-targeted two-photon fluorescence probe for the real-time ratiometric imaging of DNA damage

2016 ◽  
Vol 52 (37) ◽  
pp. 6308-6311 ◽  
Author(s):  
Hua Zhang ◽  
Kui Wang ◽  
Xiaopeng Xuan ◽  
Qingzhang Lv ◽  
Yamin Nie ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Real Time ◽  
Cancer Cell ◽  
Fluorescence Probe ◽  
The Real ◽  
Two Photon ◽  
Ratiometric Imaging ◽  
Two Photon Fluorescence ◽  
Photon Fluorescence

A sequential ICT fluorescence probe (ANF–Glu) was successfully utilized to spatially and temporally image DNA damage in cancer cells.

A nucleus targetable fluorescent probe for ratiometric imaging of endogenous NO in living cells and zebrafishes

The Analyst ◽  
2021 ◽  
Author(s):  
Liang Zhao ◽  
Zhenlong Huang ◽  
Daqing Ma ◽  
Yu Yan ◽  
Xinfu Zhang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Real Time ◽  
Fluorescent Probe ◽  
Living Cells ◽  
Live Cells ◽  
Real Time Monitoring ◽  
Immune Systems ◽  
Ratiometric Imaging ◽  
Messenger Molecule

Nitric oxide (NO) is an important cellular messenger molecule in the cardiovascular, nervous and immune systems. Real-time monitoring of NO activity in specific organelles of live cells is important to...

Applications of Flow Cytometry to Cell Adhesion Biology: From Aggregates to Drug Discovery

2005 ◽  
Author(s):  
Richard S. Larson ◽  
Alexandre Chigaev
Keyword(s):  
Flow Cytometry ◽  
Cell Adhesion ◽  
Real Time ◽  
Flow Cytometric Analysis ◽  
Target Cells ◽  
Fluid Shear ◽  
Effector Cells ◽  
Flow Cytometric ◽  
Adhesive Interactions ◽  
Over Time

Flow cytometry represents a powerful and evolving methodologic approach to cell adhesion biology. In its beginnings, flow cytometry was used solely to measure the expression of receptors on cellular surfaces and to correlate that expression with biologic function in non-flow-cytometry-based assays. From this primitive beginning, applications have proliferated and now include methodologies that measure real-time aggregation, receptor activity, and the downstream biologic consequences of cell adhesion. These biologic applications have led to platforms that are easily employed as drug screening and target validation tools. Functional assays that measure cell aggregation were initially developed to measure cell–cell interactions in the immune system, especially between cytotoxic cells and various cell types targeted as the focus of their cytotoxic activity. The cytotoxic “effector” cells and the “target” cells were stained with spectrally distinct fluorescent dyes, gently sedimented together into a cell pellet, and allowed to interact under static conditions for designated intervals of time. When resuspended and introduced into the flow cytometer, effector cells adherent to target cells were detected as “conjugate” particles emitting the fluorescence spectra of both dyes. Nonadherent effector and target cells were detected as monochromatically fluorescent particles. By using ion concentration–sensitive cytoplasmic fluorescent probes as the effector cell labels, it was also possible to detect physiological changes in intracellular ionized calcium and pH elicited by adhesion to target cells and to correlate these responses with cytotoxic function. Later, methods were developed for continuously measuring (“real-time”) cell adhesive interactions as they progressed over time in a fluid shear environment. A limitation of early adhesion kinetics analyses was that the fluid shear was generated with a magnetic stir bar and was thus neither homogeneous nor amenable to precise quantification. Subsequent refinement of these methods has enabled flow cytometric analysis of cell mixtures subjected to a more uniform and quantifiable fluid shear environment generated in a cone-plate viscometer. Cell mixtures are sampled periodically from the viscometer into a formalin fixative solution for subsequent off-line flow cytometric analysis. These experiments have been able to demonstrate a remarkable potentiation of adhesion efficiency through the combined action of two sets of adhesion molecules and a progression of adhesion molecule use from one class to another over time.

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