scholarly journals Multicolor multiphoton in vivo imaging flow cytometry

2016 ◽  
Vol 24 (6) ◽  
pp. 6126 ◽  
Author(s):  
Lingjie Kong ◽  
Jianyong Tang ◽  
Meng Cui
Keyword(s):  
Flow Cytometry ◽  
In Vivo Imaging ◽  
Imaging Flow Cytometry

In vivo Imaging Flow Cytometry of Human Leukocytes

2017 ◽  
Author(s):  
Cheng-Ham Wu ◽  
Chia-Hung Hsieh ◽  
Shih-Hung Huang ◽  
Jong-Wei Lin ◽  
Tzung-Dau Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
In Vivo Imaging ◽  
Human Leukocytes ◽  
Imaging Flow Cytometry

scholarly journals β2M Signals Monocytes Through Non-Canonical TGFβ Receptor Signal Transduction

2021 ◽  
Author(s):  
Zachary T Hilt ◽  
Preeti Maurya ◽  
Laura Tesoro ◽  
Daphne N Pariser ◽  
Sara K Ture ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Flow Cytometry ◽  
Nuclear Localization ◽  
Ubiquitin Ligase ◽  
Dependent Manner ◽  
Elevated Plasma ◽  
Imaging Flow Cytometry ◽  
Tgfβ Receptor

Rationale: Circulating monocytes can have pro-inflammatory or pro-reparative phenotypes. The endogenous signaling molecules and pathways that regulate monocyte polarization in vivo are poorly understood. We have shown that platelet derived beta-2 microglobulin (β2M) and transforming growth factor beta (TGFβ) have opposing effects on monocytes by inducing inflammatory and reparative phenotypes respectively, but each bind and signal through the same receptor. We now define the signaling pathways involved. Objective: To determine the molecular mechanisms and signal transduction pathways by which β2M and TGFβ regulate monocyte responses both in vitro and in vivo. Methods and Results: Wild-type (WT) and platelet specific β2M knockout (Plt-β2M -/- ) mice were treated intravenously with either β2M or TGFβ to increase plasma concentrations to those in cardiovascular diseases. Elevated plasma β2M increased pro-inflammatory monocytes, while increased plasma TGFβ increased pro-reparative monocytes. TGFβ receptor (TGFβR) inhibition blunted monocyte responses to both β2M and TGFβ in vivo. Using imaging flow cytometry, we found that β2M decreased monocyte SMAD2/3 nuclear localization, while TGFβ promoted SMAD nuclear translocation, but decreased non-canonical/inflammatory (JNK and NFκB nuclear localization). This was confirmed in vitro using both imaging flow cytometry and immunoblots. β2M, but not TGFβ, promoted ubiquitination of SMAD3 and SMAD4, that inhibited their nuclear trafficking. Inhibition of ubiquitin ligase activity blocked non-canonical SMAD-independent monocyte signaling and skewed monocytes towards a pro-reparative monocyte response. Conclusions: Our findings indicate that elevated plasma β2M and TGFβ dichotomously polarize monocytes. Furthermore, these immune molecules share a common receptor, but induce SMAD-dependent canonical signaling (TGFβ) versus non-canonical SMAD-independent signaling (β2M) in a ubiquitin ligase dependent manner. This work has broad implications as β2M is increased in several inflammatory conditions, while TGFβ is increased in fibrotic diseases.

scholarly journals Kinetics and Specificity of HEK293T Extracellular Vesicle Uptake using Imaging Flow Cytometry

2020 ◽  
Author(s):  
Brian Jurgielewicz ◽  
Yao Yao ◽  
Steven L. Stice
Keyword(s):  
Flow Cytometry ◽  
Genetic Material ◽  
Cell Types ◽  
Extracellular Vesicle ◽  
Human Neural Stem Cells ◽  
Imaging Flow Cytometry ◽  
Gene Therapy Vectors ◽  
Time Variables

Abstract Background : Extracellular vesicles (EVs) are nanosized vesicles naturally secreted from cells responsible for intercellular communication and delivery of proteins, lipids, and other genetic material. Ultimately, EVs could provide innate therapeutic contents and loaded therapeutic payloads such as small molecules and gene therapy vectors to recipient cells. However, comparative kinetic measures that can be used to quantify and ultimately optimize delivery and uptake of EV payloads are lacking. We investigated both dose and time effects on EV uptake and evaluated the potential specificity of EV uptake to better understand the kinetics and uptake of human embryonic kidney (HEK293T) derived EVs. Results : Utilizing an imaging flow cytometry platform (IFC), HEK293T EV uptake was analyzed. HEK293T EV uptake was dose and time dependent with a minimum threshold dose of 6,000 EVs per cell at 4 hours of co-culture. HEK293T EV uptake was inhibited when co-cultured with recipient cells at 4°C or with pre-fixed recipient cells. By co-culturing HEK293T EVs with cell lines from various germ layers, HEK293T EVs were taken up at higher quantities by HEK293T cells. Lastly, human neural stem cells (hNSCs) internalized significantly more HEK293T EVs relative to mature neurons. Conclusions : Imaging flow cytometry is a quantitative, high throughput, and versatile platform to quantify the kinetics of EV uptake. Utilizing this platform, dose and time variables have been implicated to affect EV uptake measurements making standardization of in vitro and in vivo assays vital for the translation of EVs into the clinic. In this study, we quantified the selectivity of EV uptake between a variety of cell types in vitro and found that EVs were internalized at higher quantities by cells of the same origin. The characterization of HEK293T EV uptake in vitro, notably specificity, dose response, and kinetic assays should be used to help inform and develop EV based therapeutics.

scholarly journals Development of in Vivo Models of Paediatric Burkitt Lymphoma Allowing Therapeutic Target Analysis and Drug Testing

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5501-5501
Author(s):  
Natalie Bell ◽  
Helen J Blair ◽  
Josef Vormoor ◽  
Chris Bacon ◽  
Vikki Rand ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Cell Lines ◽  
In Vivo Imaging ◽  
Target Selection ◽  
Post Transplantation ◽  
Bioluminescent Signal ◽  
Splenic Enlargement ◽  
Renal Enlargement

Abstract Childhood sporadic Burkitt lymphoma (BL) is a highly aggressive mature B-cell lymphoma. Patients require intensive chemotherapy which results in prolonged periods of hospitalisation for distressing and dangerous side effects. Additionally those with refractory disease or those that relapse have an extremely poor prognosis. As a result, there is an urgent need to develop new targeted treatments that have undergone rigorous pre-clinical evaluation. The aim of this project is to develop improved models for target selection and pre-clinical validation to reduce the failure of translation of novel agents. Two models are being developed. Firstly, xenotransplantation of BL cell lines is being used to optimise transplantation protocols and to provide an orthotopic assay for in vivo functional genomic (RNAi) screening. This is currently being used in genome-wide target identification screens. Secondly, cryopreservation of viable BL blasts is allowing development of a patient-derived xenograft model, suitable for validation of candidate targets by both RNAi and pharmacological inhibition and for testing novel therapeutic agents. The sporadic BL cell lines CA46 and RAMOS have been transplanted into NOD/LtSz-scid IL-2Rg-/- (NSG) and RAG2-/-gc-/- (RAG2) mice at reducing cell doses (105, 104 and 103 cells/mouse). This approach will identify the optimal conditions for orthotopic engraftment. Prior to transplantation, cells were transduced with the lentiviral vector pSLIEW expressing green fluorescent protein (analysis and cell sorting) and firefly luciferase (in vivo bioluminescent imaging). Engraftment was then monitored in real-time using the Caliper in vivo imaging system (IVIS). CA46 and RAMOS cell lines have been transduced with SLIEW, sorted to high purity (>90%) and engrafted in both NGS and RAG2 mice. IVIS imaging of mice transplanted with CA46 cells showed that rate of engraftment was relative to cell dose. All six mice showed a bioluminescent signal over the femurs by 3 weeks post transplantation, indicative of bone marrow (BM) engraftment. Analysis of BM by flow cytometry confirmed engraftment in the two mice analysed, as determined by GFP positivity and presence of the human B-cell markers CD10, CD19 and CD20. Renal enlargement was observed in all mice analysed, with dense infiltration by mature lymphoid blasts evident microscopically. Immunohistochemistry confirmed infiltration with human CD20 positive cells. Splenic enlargement was observed in five out of six mice and engraftment was again confirmed histologically. Onset of disease was rapid. Mice were killed when they showed signs of ill health (see table). A similar pattern of engraftment was observed in NSG and RAG2 mice transplanted with RAMOS cells, although signal intensity was not relative to transplanted cell dose. Bioluminescent signal over the femur was detected in four out of six mice by 3 weeks post transplantation. NSG and RAG2 mice receiving 103 cells, with no IVIS signal, were negative for BM engraftment by flow cytometry. Renal enlargement was not observed, however focal renal lesions were evident macroscopically in three out of six mice. These lesions were composed of human CD20 positive cells. Splenic enlargement was not observed, although a degree of splenic engraftment was identified histologically. Onset of disease was also rapid. Following the initial cell line model development, we are now able to transplant the first patient-derived BL sample. If successful, primograft material will be labelled with luciferase (pSLIEW) and re-transplanted, allowing in vivo imaging. When combined with in vivo RNAi screening, this advanced pre-clinical model for target selection and validation may help reduce target drop-out during early phase clinical trials. This is a critical step, not only to avoid exposing children to ineffectual and potentially toxic drugs, but also to avoid delay in the development of new effective therapies. Abstract 5501. TableCA46DoseSpleenKidneyBMSurvival (days)RAMOSDoseSpleenKidneyBMSurvival (days) RAG2105+++++na29 RAG2105+++++31104+++++++38104-/+-/++29103+++na38103--/+-39 NSG105++na23 NSG105-+++37104++++++31104-/+-/+na30103+++++na38103-++-39 CD20 positivity for spleen and kidney; none (-), < 10% (-/+), 10 - 50% (+), > 50% (++), 100% (+++). For BM engraftment (CD10, CD19 and CD20 positive); no engraftment (-), engrafted (+), not assessed (na). Disclosures No relevant conflicts of interest to declare.

Disruption of Wnt/β-Catenin Signaling Exerts Antileukemia Activity and Sensitizes with Tyrosine Kinase Inhibitors in FLT3 Mutated AML In Vivo

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2828-2828
Author(s):  
Xuejie Jiang ◽  
Po Yee Mak ◽  
Hong Mu ◽  
Duncan Mak ◽  
Qi Zhang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tyrosine Kinase ◽  
Survival Time ◽  
Nuclear Localization ◽  
In Vivo Imaging ◽  
Median Survival Time ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Flt3 Mutations

Abstract Wnt/β-catenin signaling is associated with pathogenesis of AML and required for establishment of leukemic stem cells. FLT3 mutations are frequently observed in AML and predict poor clinic outcomes. Aberrant activation of FLT3 signaling stabilizes β-catenin and increases its nuclear localization and transcriptional activity. FLT3 tyrosine kinase inhibitors (TKIs) are used to treat FLT3-mutated AML, but their effects are limited due to primary or secondary resistance to TKIs. We previously reported (ASH 2015) that disrupting Wnt/b-catenin signaling by C-82, a selective β-catenin/cAMP binding protein antagonist in combination with FLT3 inhibitors had a synergistic cytotoxicity in vitro in FLT3-mutated AML cells and AML stem/progenitor cells through inhibiting nuclear localization of β-catenin and suppressing the expression of β-catenin target proteins including survivin, CD44, c-Myc, and cyclin D1; several frizzled family receptors/co-receptors; Wnt ligands; and FLT3 downstream signaling proteins. The synergy was also observed in TKI-resistant FLT3 mutated cells. In this study, we evaluated the antileukemia effect of combined inhibition of β-catenin and FLT3 signaling in vivo in immunodeficient mice xenografted with FLT3-ITD mutated cells either from a cell line or from an AML patient. Molm13-GFP/Luc cells were injected into NOD-SCID IL2RγNull (NSG) mice. Once engraftment was confirmed by in vivo imaging, mice were treated with PRI-724 (C-82 pro-drug), sorafenib, or both. Treatment with PRI-724 or sorafenib decreased leukemia burden, and the combination was the most effective as assessed by in vivo imaging, flow cytometric measurement of human CD45+ cells in blood, and bone marrow (BM) and spleen H&E staining. The mice in PRI-724 (19 days, P = 0.025) or sorafenib (28 days, P = 0.0002) treated group had significantly longer median survival time than the control group (17 days), and the combined treatment further prolonged the survival time (30.5 days) (P = 0.0005, combination vs RPI-724; P = 0.0056, combination vs sorafenib). CyTOF and SPADE tree analysis showed a great reduction of human CD45+ cells and decreased expression of β-catenin, CD44, c-Myc, survivin, p-FLT3, p-ERK, p-AKT, and p-STAT5 in BM cells of the combination treated mice. Cells from a FLT3-ITD mutated AML patient sample collected from patient-derived xenograft (spleen) were injected into NOD-SCID IL2RγNull-3/GM/SF (NSGS) mice. After engraftment was confirmed by flow cytometry, mice were treated as above. Leukemia burden was decreased by sorafenib or PRI-724 treatment, as determined by flow cytometry measurement of human CD45+ cells in blood, BM, and spleen samples, but did not reach statistical significance. The combination significantly enhanced the antileukemia effect. PRI-724 (31 days, P = 0.008) or sorafenib (48 days, P = 0.0003) significantly prolonged median survival time compared with control (29 days), and the combination further extended the survival time (54 days) (P = 0.0005, combination vs RPI-724; P = 0.0067, combination vs sorafenib). Our in vivo study further demonstrates that disruption of Wnt/b-catenin signaling exerts antileukemia activity and sensitizes with TKIs in FLT3 mutated AML. These findings provide a rationale for clinic development of combined inhibition of Wnt/β-catenin and FLT3 signaling to overcome resistance and improve outcomes in AML patients with FLT3 mutations. Disclosures Konopleva: Cellectis: Research Funding; Calithera: Research Funding. Carter:PRISM Pharma/Eisai: Research Funding.

scholarly journals HKOCl-3: a fluorescent hypochlorous acid probe for live-cell and in vivo imaging and quantitative application in flow cytometry and a 96-well microplate assay

2016 ◽  
Vol 7 (3) ◽  
pp. 2094-2099 ◽  
Author(s):  
Jun Jacob Hu ◽  
Nai-Kei Wong ◽  
Ming-Yang Lu ◽  
Xingmiao Chen ◽  
Sen Ye ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Fluorescent Probe ◽  
In Vivo Imaging ◽  
Hypochlorous Acid ◽  
Live Cell ◽  
Microplate Assay

We describe an ultra-selective and ultra-sensitive HOCI fluorescent probe HKOCl-3 for live-cell and in vivo imaging and quantitative applications.

Multiple dye-doped NIR-emitting silica nanoparticles for both flow cytometry and in vivo imaging

RSC Advances ◽  
2014 ◽  
Vol 4 (35) ◽  
pp. 18278-18285 ◽  
Author(s):  
Stefania Biffi ◽  
Luca Petrizza ◽  
Enrico Rampazzo ◽  
Rebecca Voltan ◽  
Massimo Sgarzi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Silica Nanoparticles ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Toxic Materials

Dye-doped near infrared-emitting silica nanoparticles (DD-NIRsiNPs) represent a valuable tool in bioimaging, because they provide sufficient brightness, resistance to photobleaching and consist of hydrophilic non-toxic materials.

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