Highly Multiplexed Phenotypic Imaging for Cell Proliferation Studies

Cappella, Paolo; Gasparri, Fabio

doi:10.1177/1087057113495712

Cited by 16 publications

(7 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cells were fixed in 2% paraformaldehyde for 30 minutes and stained with Hoechst 33342 (10 µg/ml, Sigma‐Aldrich) for 1 hour at room temperature. Cell cycle analysis was performed using a high‐content screening platform (ArrayScan VTI) by scanning 20 fields/well using the Cell Cycle Bioapplication V3 software (Thermo Scientific) as described previously .…”

Section: Methodsmentioning

confidence: 99%

YB-1 Transforms Human Mammary Epithelial Cells Through Chromatin Remodeling Leading to the Development of Basal-Like Breast Cancer

et al. 2014

View full text Add to dashboard Cite

There is growing evidence that cancer-initiation could result from epigenetic changes. Y-box binding protein-1 (YB-1) is a transcription/translation factor that promotes the formation of tumors in transgenic mice; however, the underlying molecular events are not understood. To explore this in a human model system, YB-1 was expressed in mammary epithelial cells under the control of a tetracycline-inducible promoter. The induction of YB-1 promoted phenotypes associated with malignancy in three-dimensional breast acini cultures. This was attributed to YB-1 enhancing the expression and activity of the histone acetyltransferase p300 leading to chromatin remodeling. Specifically, this relaxation of chromatin allowed YB-1 to bind to the BMI1 promoter. The induction of BMI1 engaged the Polycomb complex resulting in histone H2A ubiquitylation and repression of the CDKN2A locus. These events manifested functionally as enhanced self-renewal capacity that occurred in a BMI1-dependent manner. Conversely, p300 inhibition with anacardic acid prevented YB-1 from binding to the BMI1 promoter and thereby subverted self-renewal. Despite these early changes, full malignant transformation was not achieved until RSK2 became overexpressed concomitant with elevated hTERT activity. The YB-1/RSK2/hTERT expressing cells formed tumors in mice that were molecularly subtyped as basal-like breast cancer. We conclude that YB-1 cooperates with p300 to allow BMI1 to over-ride p16INK4a-mediated cell cycle arrest enabling self-renewal and the development of aggressive breast tumors.

show abstract

Section: Methodsmentioning

confidence: 99%

YB-1 Transforms Human Mammary Epithelial Cells Through Chromatin Remodeling Leading to the Development of Basal-Like Breast Cancer

et al. 2014

View full text Add to dashboard Cite

show abstract

“…EdU in combination with BrdU azide derivatives can also be used for multiparametric analysis by high-content screening (HCS) readers (Cappella et al, 2008;Cappella and Gasparri, 2014), e.g., an ArrayScan VTI reader (ThermoFisher Scientific).…”

Section: Fluorescence Immunocytochemistry and Image Acquisition By Himentioning

confidence: 99%

Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining

et al. 2015

Self Cite

View full text Add to dashboard Cite

Determination of incorporation of the thymidine analog 5‐bromo‐2′‐deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle. However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S‐phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by 5‐ethynyl‐2′‐deoxyuridine (EdU) which is incorporated into DNA. The nucleotide‐exposed ethynyl residue was then derivatized by a copper‐catalyzed cycloaddition reaction (“click chemistry” coupling) using a BrdU azide probe. The resulting DNA‐bound bromouracil moieties were then detected by commercial anti‐BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is more sensitive than traditional BrdU and allows multicolor and multiplex analysis in flow cytometry (FCM) and image‐based cytometry. © 2015 by John Wiley & Sons, Inc.

show abstract

“…Analysis of nuclear morphology can provide valuable insights into a cell’s state, such as cell cycle phase or cell health 42 43 44 . Here, we demonstrate how to conduct experiments to track nuclear morphology over extended periods of time and test the limitations of the assay and screen for phototoxic events.…”

Section: Resultsmentioning

confidence: 99%

Single cell dynamic phenotyping

Patsch

Chiu

Engeln

et al. 2016

Sci Rep

View full text Add to dashboard Cite

Live cell imaging has improved our ability to measure phenotypic heterogeneity. However, bottlenecks in imaging and image processing often make it difficult to differentiate interesting biological behavior from technical artifact. Thus there is a need for new methods that improve data quality without sacrificing throughput. Here we present a 3-step workflow to improve dynamic phenotype measurements of heterogeneous cell populations. We provide guidelines for image acquisition, phenotype tracking, and data filtering to remove erroneous cell tracks using the novel Tracking Aberration Measure (TrAM). Our workflow is broadly applicable across imaging platforms and analysis software. By applying this workflow to cancer cell assays, we reduced aberrant cell track prevalence from 17% to 2%. The cost of this improvement was removing 15% of the well-tracked cells. This enabled detection of significant motility differences between cell lines. Similarly, we avoided detecting a false change in translocation kinetics by eliminating the true cause: varied proportions of unresponsive cells. Finally, by systematically seeking heterogeneous behaviors, we detected subpopulations that otherwise could have been missed, including early apoptotic events and pre-mitotic cells. We provide optimized protocols for specific applications and step-by-step guidelines for adapting them to a variety of biological systems.

show abstract

Highly Multiplexed Phenotypic Imaging for Cell Proliferation Studies

Cited by 16 publications

References 41 publications

YB-1 Transforms Human Mammary Epithelial Cells Through Chromatin Remodeling Leading to the Development of Basal-Like Breast Cancer

YB-1 Transforms Human Mammary Epithelial Cells Through Chromatin Remodeling Leading to the Development of Basal-Like Breast Cancer

Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining

Single cell dynamic phenotyping

Contact Info

Product

Resources

About