List of Titles

Apoptosis and beyond: cytometry in studies of programmed cell death

- Wlodkowic, Donald, Telford, W, Skommer, Joanna, Darzynkiewicz, Zbigniew

Authors: Wlodkowic, Donald , Telford, W , Skommer, Joanna , Darzynkiewicz, Zbigniew
Date: 2011
Type: Text , Book chapter
Relation: Methods in Cell Biology p. 55-98
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Description: A cell undergoing apoptosis demonstrates multitude of characteristic morphological and biochemical features, which vary depending on the inducer of apoptosis, cell type and the “time window” at which the process of apoptosis is observed. Because the gross majority of apoptotic hallmarks can be revealed by flow and image cytometry, the cytometric methods become a technology of choice in diverse studies of cellular demise. Variety of cytometric methods designed to identify apoptotic cells, detect particular events of apoptosis and probe mechanisms associated with this mode of cell death have been developed during the past two decades. In the present review, we outline commonly used methods that are based on the assessment of mitochondrial transmembrane potential, activation of caspases, DNA fragmentation, and plasma membrane alterations. We also present novel developments in the field such as the use of cyanine SYTO and TO-PRO family of probes. Strategies of selecting the optimal multiparameter approaches, as well as potential difficulties in the experimental procedures, are thoroughly summarized

Analysis of individual molecular events of DNA damage response by flow- and image-assisted cytometry

- Darzynkiewicz, Zbigniew, Traqanos, Frank, Zhao, Hong, Halicka, H Dorota, Skommer, Joanna, Wlodkowic, Donald

Authors: Darzynkiewicz, Zbigniew , Traqanos, Frank , Zhao, Hong , Halicka, H Dorota , Skommer, Joanna , Wlodkowic, Donald
Date: 2011
Type: Text , Book chapter
Relation: Methods in Cell Biology p. 115-147
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Description: This chapter describes molecular mechanisms of DNA damage response (DDR) and presents flow- and image-assisted cytometric approaches to assess these mechanisms and measure the extent of DDR in individual cells. DNA damage was induced by cell treatment with oxidizing agents, UV light, DNA topoisomerase I or II inhibitors, cisplatin, tobacco smoke, and by exogenous and endogenous oxidants. Chromatin relaxation (decondensation) is an early event of DDR chromatin that involves modification of high mobility group proteins (HMGs) and histone H1 and was detected by cytometry by analysis of the susceptibility of DNA in situ to denaturation using the metachromatic fluorochrome acridine orange. Translocation of the MRN complex consisting of Meiotic Recombination 11 Homolog A (Mre11), Rad50 homolog, and Nijmegen Breakage Syndrome 1 (NMR1) into DNA damage sites was assessed by laser scanning cytometry as the increase in the intensity of maximal pixel as well as integral value of Mre11 immunofluorescence. Examples of cytometric detection of activation of Ataxia telangiectasia mutated (ATM), and Check 2 (Chk2) protein kinases using phospho-specific Abs targeting Ser1981 and Thr68 of these proteins, respectively are also presented. We also discuss approaches to correlate activation of ATM and Chk2 with phosphorylation of p53 on Ser15 and histone H2AX on Ser139 as well as with cell cycle position and DNA replication. The capability of laser scanning cytometry to quantify individual foci of phosphorylated H2AX and/or ATM that provides more dependable assessment of the presence of DNA double-strand breaks is outlined. The new microfluidic Lab-on-a-Chip platforms for interrogation of individual cells offer a novel approach for DDR cytometric analysis.

Multivariate analysis of apoptotic markers versus cell cycle phase in living human cancer cells by microfluidic cytometry

- Akagi, Jin, Skommer, Joanna, Matuszek, Anna, Takeda, Kazuo, Fujimura, Yuu, Khoshmanesh, Khashayar, Kalantar-zadeh, Kourosh, Mitchell, Arnan, Errington, Rachel, Smith, Paul, Darzynkiewicz, Zbigniew, Wlodkowic, Donald

Authors: Akagi, Jin , Skommer, Joanna , Matuszek, Anna , Takeda, Kazuo , Fujimura, Yuu , Khoshmanesh, Khashayar , Kalantar-zadeh, Kourosh , Mitchell, Arnan , Errington, Rachel , Smith, Paul , Darzynkiewicz, Zbigniew , Wlodkowic, Donald
Date: 2013
Type: Text , Conference paper
Relation: Microfluidics, BioMEMS, and Medical Microsystems XI
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Description: Measurement of apoptotic markers in tumors can be directly correlated with the cell cycle phase using flow cytometry (FCM). The conventional DNA content analysis requires cell permeabilization to stain nuclei with fluorescent probes such as propidium iodide or use of a costly UV-excitation line for Hoechst 33342 probe. The access to FCM is also still limited to centralized core facilities due to its inherent high costs and complex operation. This work describes development and proof-of-concept validation of a portable and user-friendly microfluidic flow cytometer (μFCM) that can perform multivariate real time analysis on live cells using sampling volumes as small as 10 microliters. The μFCM system employs disposable microfluidic cartridges fabricated using injection molding in poly(methylmethacrylate) transparent thermoplastic. Furthermore, the dedicated and miniaturized electronic hardware interface enables up to six parameter detection using a combination of spatially separated solid-state 473 (10 mW) and 640 nm (20 mW) lasers and x-y stage for rapid laser alignment adjustment. We provide new evidence that a simple 2D flow focusing on a chip is sufficient to measure cellular DNA content in live tumor cells using a far-red DNA probe DRAQ5. The feasibility of using the μFCM system for a dose-response profiling of investigational anti-cancer agents on human hematopoietic cancer cells is also demonstrated. The data show that μFCM can provide a viable novel alternative to conventional FCM for multiparameter detection of caspase activation and dissipation of mitochondrial inner membrane potential (ΔΨm) in relation to DNA content (cell cycle phase) in live tumor cells.
Description: Measurement of apoptotic markers in tumors can be directly correlated with the cell cycle phase using flow cytometry (FCM). The conventional DNA content analysis requires cell permeabilization to stain nuclei with fluorescent probes such as propidium iodide or use of a costly UV-excitation line for Hoechst 33342 probe. The access to FCM is also still limited to centralized core facilities due to its inherent high costs and complex operation. This work describes development and proof-of-concept validation of a portable and user-friendly microfluidic flow cytometer (

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