Live Cell Insights Publications Newsletter

Vol. 4, 2018 — Featured publications for the IncuCyte® Live-Cell Analysis System

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2,000 publications and counting!

IncuCyte has reached 2,000 cited publications spanning a wide array of research areas and applications. We’ve experienced over 50% growth in publications in just the last year! Search our publications list to see what exciting research is being published using the IncuCyte® Live-Cell Analysis System.

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Genome-wide CRISPR screens in primary human T Cells reveal key regulators of immune function

New genome-wide CRISPR screening platform in primary human T cells

Cytotoxic T cells play an immunomodulatory role in controlling human diseases, including cancer. CRISPR-based functional studies in T cells are useful to cancer drug development, but are difficult to carry out in human primary cells. In a joint study, scientists in the laboratory of Dr. Alexander Marson from the University of California, San Francisco, developed a new method, single guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE), which enables loss-of-function pooled screening in primary human T cells at genome-wide scale. Key findings include:

  • The SLICE method was used to identify gene targets that regulate T cell proliferation in response to T cell receptor (TCR) stimulation.
  • High scoring hits were confirmed for their ability to affect TCR response using arrayed editing with Cas9 RNPs.
  • Screening hits induced caspase-mediated cell death in engineered T cells, as measure on the IncuCyte® Live-Cell Analysis System.

Read the full paper in Cell, November 2018.



IncuCyte® Apoptosis Assays

The mix-and-read IncuCyte® Caspase-3/7 and Annexin V Reagents let you measure multiple apoptotic pathways simultaneously and in real time...

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Cell Biology

Nonlinear relationship between ER Ca2+ depletion versus induction of the unfolded protein response, autophagy inhibition, and cell death

Extreme Ca2+ depletion triggers UPR response and cell death

Endoplasmic reticulum (ER) Ca2+ depletion acts as a stress signal to activate the unfolded protein response (UPR), which in turn engages the autophagy and cell death pathways. However, the relationship between these events and the extent and duration of ER Ca2+ depletion is not fully understood. A study led by Dr. Paula Szalaia at the Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership for Molecular Medicine, University of Oslo, Norway, investigated this question by gradually depleting Ca2+ in PC3 cells using sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors.

Their data showed that UPR induction, autophagy inhibition or cell death occur in response to extreme Ca2+ depletion. Key findings include:

  • Increasing concentrations of SERCA inhibitors resulted in cell death and growth inhibition in a dose-dependent manner. However, the cells tolerated strong and sustained Ca2+ depletion without UPR, activation, cell death, or inhibition of bulk autophagy.
  • Similar results were observed under extreme Ca2+ depletion, where cell proliferation was reduced and Ca2+ was not detectable even in the presence of purging agents.
  • Ca2+ depletion using extracellular EGTA produced similar results, where extreme depletion was necessary to induce UPR activation and cell death.
  • The IncuCyte® Live-Cell Analysis System was used to perform direct ER Ca2+ measurements and assessment of cell death and confluency.

Read the full paper in Cell Calcium, December 2018.



IncuCyte® Proliferation Assays

Measure cell proliferation using live cell time-lapse imaging, with or without labels. Easily generate long-term growth and growth-inhibition curves and monitor morphology.

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Three-dimensional induction of dorsal, intermediate and ventral spinal cord tissues from human pluripotent stem cells

A tool for analyzing human spinal cord development

The spinal cord has well-organized neural circuits that sense the environment and produce the appropriate motor response. Despite in vitro data describing motor neuron generation, there are no reports that show induction of the whole spinal cord tissue. In a joint study led by Dr. Takenori Ogura at the Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Japan, and Dr. Hideya Sakaguchi at the Department of Neurosurgery, Kyoto University Graduate School of Medicine, Japan, scientists achieved three dimensional induction of dorsal spinal cord-like tissues using human pluripotent stem cells (hPSCs).

Using this technique, they successfully induced dorsal, intermediate and ventral spinal cord-like tissues. Key findings include:

  • A previously-described protocol for in vitro spinal motor neuron induction was modified to include a new 3-dimensional spinal cord (3-DiSC) condition. The IncuCyte® S3 Spheroid Software Module was used to monitor cell reaggregation during the initial seeding of human induced pluripotent cells.
  • The 3-DiSC condition allowed for generating four distinct dorsal interneuron marker-positive cell populations, as confirmed by quantitative PCR and immunohistochemistry for the expression of relevant markers.
  • Treatment with bone morphogenetic proteins (BMPs) dorsalized the character of the interneurons, while treatment with sonic hedgehog (Shh) induced intermediate and ventral spinal cord-like tissues. These tissues could form several spinal neurons in vitro.

Read the full paper in Development, July 2018.



IncuCyte® S3 Spheroid Software Module

Analyze growth and shrinkage of spheroids with or without fluorescent label in multi-well formats on the IncuCyte® Live-Cell Analysis System.

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Preparation, characterization, and in vivo evaluation of an oral multiple nanoemulsive system for co-delivery of pemetrexed and quercetin

An oral co-delivery system with improved absorption

Co-administration of anticancer drugs with natural chemotherapeutic agents offers anticancer efficacy with reduced adverse effects. In a joint study between the College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Korea, and the College of Pharmacy, Pusan National University, Korea, scientists designed an oral co-delivery system for IV cancer drug pemetrexed (PMX) and quercetin (QCN), an abundant natural flavonoid.

Their water-in-oil-in-water (w/o/w) nanoemulsion (NE) system showed synergistic efficacy and improved oral absorption in vivo. Key findings include:

  • To improve permeability, PMX was ion paired with Nα-deoxycholyl-L-lysyl-methylester (DCK) and incorporated into the inner aqueous phase of a w/o/w multiple NE. QCN was loaded into the oil phase of an NE. Membrane permeability across an artificial and Caco-2 cell monolayer was improved for both compounds.
  • Cell viability assays on A549 cells showed synergistic cytotoxic effects of PMX or PMX/DCK in combination with QCN, with different anticancer activity. Live-cell fluorescence images of caspase-3/7 activity captured on the IncuCyte® system indicated significant apoptotic activity after treatment with PMX/DCK and QCN. A would healing assay performed on the IncuCyte system also revealed an improved synergistic inhibition of cell proliferation/migration.
  • The NE formulation showed improved oral bioavailability in rats and a maximal tumor growth suppression in orally-treated A549 cell-bearing mice.

Read the full paper in Pharmaceutics, September 2018.



IncuCyte® Caspase-3/7 Green Apoptosis Assay Reagent

The IncuCyte® Caspase-3/7 Green Apoptosis Reagent couples the activated caspase-3/7 recognition motif (DEVD) to NucView™ 488, a DNA intercalating dye to enable quantification of apoptosis over time.

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Kunitz type protease inhibitor EgKI-1 from the canine tapeworm Echinococcus granulosus as a promising therapeutic against breast cancer

Anti-cancer properties of EgKI-1

EgKI-1 protease is highly expressed by the infectious oncosphere stage of the canine tapeworm Echinococcus granulosus, which causes hydatid cysts in human organs. Larval protoscoleces inside the hydatid cysts have demonstrated killing of some tumors via an unknown mechanism. A study led by Dr. Shiwanthi Ranasinghe at the Molecular Parasitology Laboratory, Immunology Department, QIMR Berghofer Medical Research Institute, Australia, showed antitumor activity of recombinant EgKI-1 protease in several cancer cell lines. They also found in vivo evidence of tumor growth inhibition in an EgKI-1-treated breast cancer mouse model. Key findings include:

  • In end point growth assays using sulforhodamine B and real-time monitoring on the IncuCyte® system, treatment with recombinant EgKI-1 inhibited the growth of breast, melanoma, cervical, and other cancer cells in a dose-dependent manner. EgKI-1 treatment also inhibited cell migration of cancer cell lines in a real-time scratch wound assay on the IncuCyte system.
  • Cell cycle profiling of MDA-MB-231 and HeLa cells indicated that EgKI-1 treatment disrupts the cell cycle. Flow cytometry analysis revealed that EgKI-1 causes apoptosis of breast cancer cells as measured using an Annexin V assay.
  • In vivo studies in triple negative breast cancer (MDA-MB-231) in BALB/c nude mice showed over 50% reduced tumor growth after 26 days of EgKI-1 treatment.

Read the full paper in PLoS One, August 2018.



IncuCyte® Cell Migration Kit

Comprises everything you need to run fully kinetic and automated 96-well cell migration and invasion scratch wound assay.

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Real-time vital mineralization detection and quantification during in vitro osteoblast differentiation

A robust, live-cell alternative to Alizarin Red staining

Alizarin Red is traditionally used to detect mineralization as a marker of osteoblast differentiation. However, the requirement of cell fixation limits this method to one time point per sample. Dr. Anastassia Serguienko at the Institute for Cancer Research, Oslo University Hospital, Norway, and colleagues, addressed this technical gap by developing a live-cell imaging methodology using Calcein Green.

They combined Calcein Green and the IncuCyte® system as a live-cell imaging tool for monitoring the dynamics of matrix mineralization. Key findings include:

  • Primary mesenchymal stem cells were differentiated into osteoblasts in the presence of Calcein Green and mineralization dynamics were monitored and quantified on the IncuCyte® Live-Cell Analysis System. Osteoblast mineralization was accurately captured in real time over the differentiation period.
  • This method avoids the drawback of the Alizarin Red assay, namely the single time point limitation, inadequate sensitivity and high background. The robust signal and convenience of the Calcein Green/ IncuCyte® method makes it the ideal way to assess bone mineralization in vitro.

Read the full paper in Methodology, August 2018.



IncuCyte® S3 Live-Cell Analysis System

The IncuCyte® S3 Live-Cell Analysis System automatically acquires and analyzes images around the clock, providing information-rich analysis that is easy to achieve.

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Mebendazole stimulates CD14+ myeloid cells to enhance T-cell activation and tumour cell killing

The immune stimulatory and anticancer effects of mebendazole

Mebendazole (MBZ), used to treat helminthic diseases, has demonstrated anticancer activity and induced a proinflammatory tumour-suppressive M1 phenotype in THP-1 monocytes and macrophages. Dr. Jenny Rubin and colleagues at the departments of Medical Sciences and Immunology, Uppsala University, Sweden, used human peripheral blood mononuclear cells (PBMCs) co-cultured with tumor cells to understand the immune modulating and anticancer properties of MBZ.

They found that MBZ potentiated the immune stimulatory and anticancer activity of CD3/IL2-activated PBMCs and that this effect was dependent on the presence of attenuated CD14 positive cells. Key findings include:

  • Screening using the Biomap platform looked for MBX-induced biomarkers in co-cultures of T-cell receptor-activated PBMCs, HT29 colon cancer cells and either human fibroblasts or human umbilical vein endothelial cells (HUVEC) cells. An immuno-stimulatory activity was observed as indicated by a significant increase in TNFα and IFNγ.
  • PBMCs activated by CD3/IL2 stimulation and MBZ showed increased PBMC clustering on the IncuCyte® system and release of pro-inflammatory IFNγ, TNFα, IL6 and IL1β cytokines.
  • In tumor cell killing assays performed on the IncuCyte® system MBZ-treated PBMC cultures had enhanced cell killing of lung cancer cells in a manner that was dependent on the presence of CD14 monocytes/macrophages.

Read the full paper in Oncotarget, July 2018.



IncuCyte® Immune Cell Killing Assays

An integrated solution for real-time visualization and automated analysis of immune cell-mediated killing of tumor cells – all within your tissue culture incubator...

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Infectious Disease

Endothelial colony-forming cell function is reduced during HIV infection

A mechanism for increased cardiovascular disease risk in HIV patients

Systemic inflammation is believed to cause an increased risk of cardiovascular disease (CVD) in human immunodeficiency virus (HIV) patients, but the mechanism remains unknown. A study lead by Dr. Samir Gupta at the Indiana University School of Medicine, Indianapolis, used a new flow cytometry assay to study how monocyte activation leads to CVD in HIV patients, including those receiving suppressive antiretroviral therapy (ART).

The data showed that monocyte activation in HIV-infected individuals adversely affects the functional parameters of endothelial progenitor cells, “endothelial colony-forming cells” (ECFCs), which have angiogenetic properties. Key findings include:

  • Flow cytometry analysis of blood samples from HIV-negative (uninfected group), HIV-positive participants not receiving ART (infected, untreated group), and HIV-positive participants receiving ART (infected, treated group) showed no differences in ECFC percentages. However, the infected, untreated participants had lower values for capillary proliferative capacity parameters, compared to values for uninfected participants or infected, treated participants.
  • Live-cell imaging analysis on the IncuCyte® system was used to measure the proliferative capacity of cord blood ECFC. The results showed reduced capillary network formation in the uninfected group as compared to the infected groups.
  • Flow cytometry studies revealed no difference in the three monocyte subsets. However, the HIV-positive groups showed a negative correlation between CD14+CD16+ intermediate monocytes and soluble CD163 and several plasma-treated, cord blood ECFC proliferative capacity parameters.

Read the full paper in The Journal of Infectious Diseases, September 2018.



IncuCyte® S3 Live-Cell Analysis System

The IncuCyte® S3 Live-Cell Analysis System automatically acquires and analyzes images around the clock, providing information-rich analysis that is easy to achieve.

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