Evaluation of membrane physiology following fluorescence activated or magnetic cell separation

Seidl, J.; Knuechel, Ruth; Kunz‐Schughart, Leoni A.

doi:10.1002/(sici)1097-0320(19990601)36:2<102::aid-cyto3>3.3.co;2-4

Cited by 37 publications

(37 citation statements)

References 24 publications

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“…This might damage the rare sorted cells and could have a negative influence on cell survival (149,150). However, the high sorting speed of up to 100,000 events per second and the ability to sort different populations in parallel make this technique well suited for the sorting of rare events like stem cells.…”

Section: Review Articlesupporting

confidence: 73%

Flow cytometry in cancer stem cell analysis and separation

et al. 2012

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In recent years, a special type of cancer cell-the cancer stem cell (CSC)-has been identified and characterized for different tumors. CSCs may be responsible for the recurrence of a tumor following a primarily successful therapy and are thought to bear a high metastatic potential. For the development of efficient treatment strategies, the establishment of reliable methods for the identification and effective isolation of CSCs is imperative. Similar to their stem cell counterparts in bone marrow or small intestine, different cluster of differentiation surface antigens have been characterized, thus enabling researchers to identify them within the tumor bulk and to determine their degree of differentiation. In addition, functional properties characteristic of stem cells can be measured. Side population analysis is based on the stem cell-specific activity of certain ATP-binding cassette transporter proteins, which are able to transport fluorescent dyes out of the cells. Furthermore, the stem cell-specific presence of aldehyde dehydrogenase isoform 1 can be used for CSC labeling. However, the flow cytometric analysis of these CSC functional features requires specific technical adjustments. This review focuses on the principles and strategies of the flow cytometric analysis of CSCs and provides an overview of current protocols as well as technical requirements and pitfalls. A special focus is set on side population analysis and analysis of ALDH activity. Flow cytometrybased sorting principles and future flow cytometric applications for CSC analysis are also discussed. ' 2012 International Society for Advancement of Cytometry

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Section: Review Articlesupporting

confidence: 73%

Flow cytometry in cancer stem cell analysis and separation

et al. 2012

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“…can cause cell damage (Seidl et al, 1999). A method that overcomes some of these limitations is impedance spectroscopy, also referred to as dielectric spectroscopy.…”

Section: A U T H O R ' S P E R S O N a L C O P Ymentioning

confidence: 99%

Label-free detection of Babesia bovis infected red blood cells using impedance spectroscopy on a microfabricated flow cytometer

et al. 2007

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Impedance spectroscopy is a powerful tool for label-free analysis and characterisation of living cells. In this work, we achieved the detection of Babesia bovis infected red blood cells using impedance spectroscopy on a microfabricated flow cytometer. The cellular modifications caused by the intracellular parasite result in a shift in impedance which can be measured dielectrically. Thus, a rapid cell-by-cell detection with microliter amounts of reagents is possible. Unlike other diagnostic tests, this method does not depend on extensive sample pre-treatment or expensive chemicals and equipment.

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“…Sort regions were defined in the forward scatter vs. FL-1 fluorescence dot blot diagram, as described. 12 For MACS, the VarioMACS system (Miltenyi Biotec, Bergisch Gladbach, Germany) was applied using a positive selection protocol with RS ϩ columns. Fibroblasts were labeled with antifibroblast microbeads (Miltenyi Biotec) according to the manufacturer's instructions.…”

Section: Cell Sortingmentioning

confidence: 99%

Three‐dimensional fibroblast–tumor cell interaction causes downregulation of RACK1 mRNA expression in breast cancer cells in vitro

Seidl

Huettinger

Knuechel

et al. 2002

Intl Journal of Cancer

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The complex cellular tumor microenvironment comprises various host cell types, e.g., endothelial, immune-competent and inflammatory cells as well as fibroblasts. Indeed, fibroblasts are the most abundant stromal cell type in desmoplastic tumors. Tumorassociated desmoplasia is a fibrotic host response manifested by an accumulation of tumor-associated stromal fibroblasts (TAFs) and ECM deposition. Manifestation and degree of the desmoplastic reaction are highly variable even within one tumor type. It is most frequently described in squamous cell carcinomas, biliopancreatic carcinomas as well as adenocarcinomas of the breast and ovaries, gastrointestinal tract and lung, excluding small cell lung cancers. Indeed, the majority of invasive ductal breast tumors develop a pronounced desmoplastic reaction, indicating that this stromal compartment not only has a profound impact on tumor cell behavior but should also be considered as a therapeutic target.Over the past 15 years, TAFs have been shown to exhibit an abnormal spatiotemporal phenotype with a multitude of modifications in the expression pattern compared to normal fibroblasts. They were identified as active participants and modulators of tumor growth and dissemination. TAFs may, e.g., exhibit myofibroblastic or fetal-like phenotypes accompanied by synthesis of intracellular smooth muscle markers and expression of so-called oncofetal ECM components such as the ED-A fibronectin splice variant. The altered expression profile also includes ECM-modulating factors such as matrix metalloproteinases, peptide growth factors and chemokines/cytokines. 1,2 However, the intricate network of bidirectional interactions between tumor cells and fibroblasts remains incompletely understood. [3][4][5][6] Previously, we established and characterized a 3-D coculture system that allows investigation of tumor cell-fibroblast interactions in a well-defined 3-D environment. 7 Here, the initial bladder cancer-fibroblast model system 8 is not only modified by the application of different breast tumor cell lines grown as spheroids but also extended to the use of fibroblasts outgrown from breast tumor biopsies. The system reflects the in vivo situation with regard to tumor cell infiltration/invasion capacity and fibroblast differentiation.Our aim was to gain deeper insight into the reciprocal interactive processes between breast tumor cells and fibroblasts, to eventually define novel therapeutic targets. To identify genes in tumor cells that may be differentially regulated following contact with fibroblasts, 3 breast tumor cell lines (BT474, T47D, MCF-7) with different invasive and myofibroblast-inducing potential in tumorfibroblast spheroid coculture were investigated. The technique of breast tumor-fibroblast spheroid coculturing 7 was combined with MACS and FACS technologies and RAP-PCR, according to Differences in the mRNA levels of tumor cells resulting from their interaction with fibroblasts were verified by specific RT-PCR and reverse Northern blotting. MATERIAL AND METHODS Cell culture...

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Evaluation of membrane physiology following fluorescence activated or magnetic cell separation

Cited by 37 publications

References 24 publications

Flow cytometry in cancer stem cell analysis and separation

Flow cytometry in cancer stem cell analysis and separation

Label-free detection of Babesia bovis infected red blood cells using impedance spectroscopy on a microfabricated flow cytometer

Three‐dimensional fibroblast–tumor cell interaction causes downregulation of RACK1 mRNA expression in breast cancer cells in vitro

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