Use of a mechanical dissociation device to improve standardization of flow cytometric cytokeratin DNA measurements of colon carcinomas

Brockhoff, Gero; Fleischmann, Sonja; Meier, Anke; Wachs, Frank-Peter; Hofstaedter, Ferdinand; Knuechel, Ruth

doi:10.1002/(sici)1097-0320(19990815)38:4<184::aid-cyto5>3.0.co;2-w

Cited by 25 publications

(34 citation statements)

References 33 publications

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“…After intra-operative confirmation of a diagnosis of GBM, fresh tissue samples were obtained by biopsy or resection of tumour. Samples were mechanically disaggregated using the Medimachine (DAKO Diagnostika GmbH, Hamburg, Germany) (Brockhoff et al, 1999) and introduced into pre-warmed DMEM (Life Technologies, Grand Island, NY, USA) containing 10% FBS (CSL, Melbourne, VIC, Australia), 2 mM glutamine (Sigma Chemical Co, St Louis, MO, USA) and 50 U ml À1 penicillin/ 50 mg ml À1 streptomycin, respectively (Life Technologies). After 24 h, any non-adherent cells and material were discarded and the media replenished.…”

Section: Methodsmentioning

confidence: 99%

In vitro sensitivity testing of minimally passaged and uncultured gliomas with TRAIL and/or chemotherapy drugs

et al. 2008

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TRAIL/Apo-2L has shown promise as an anti-glioma drug, based on investigations of TRAIL sensitivity in established glioma cell lines, but it is not known how accurately TRAIL signalling pathways of glioma cells in vivo are reproduced in these cell lines in vitro. To replicate as closely as possible the in vivo behaviour of malignant glioma cells, 17 early passage glioma cell lines and 5 freshly resected gliomas were exposed to TRAIL-based agents and/or chemotherapeutic drugs. Normal human hepatocytes and astrocytes and established glioma cell lines were also tested. Cross-linked TRAIL, but not soluble TRAIL, killed both normal cell types and cells from three tumours. Cells from only one glioma were killed by soluble TRAIL, although only inefficiently. High concentrations of cisplatin were lethal to glioma cells, hepatocytes and astrocytes. Isolated combinations of TRAIL and chemotherapy drugs were more toxic to particular gliomas than normal cells, but no combination was generally selective for glioma cells. This study highlights the widespread resistance of glioma cells to TRAIL-based agents, but suggests that a minority of high-grade glioma patients may benefit from particular combinations of TRAIL and chemotherapy drugs. In vitro sensitivity assays may help identify effective drug combinations for individual glioma patients.

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Section: Methodsmentioning

confidence: 99%

In vitro sensitivity testing of minimally passaged and uncultured gliomas with TRAIL and/or chemotherapy drugs

et al. 2008

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“…Dissociation of small tissue pieces (a few cubic millimeters each) of fresh-frozen paired myocardial samples was performed as previously published. 13 Disaggregated cells were indirectly stained anti-MEF2 (clone 17785, 1:10, Santa Cruz Biotechnology, Inc, Santa Cruz, Calif; fluorescein isothiocyanateconjugated secondary antibody, 1:20, Dako GmbH, Hamburg, Germany). Samples were measured on a FACSCanto-II analyzer (BD Biosciences, Heidelberg, Germany).…”

Section: Flow Cytometry-based Evaluation Of Cardiomyocyte Dna Contentmentioning

confidence: 99%

Hemodynamic Support by Left Ventricular Assist Devices Reduces Cardiomyocyte DNA Content in the Failing Human Heart

et al. 2010

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Background-Whether adult cardiomyocytes have the capacity to regenerate in response to injury and, if so, to what extent are still issues of intense debate. In human heart failure, cardiomyocytes harbor a polyploid genome. A unique opportunity to study the mechanism of polyploidization is provided through the setting of hemodynamic support by left ventricular assist devices. Hence, the cardiomyocyte DNA content, nuclear morphology, and number of nuclei per cell were assessed before and after left ventricular assist device support. Methods and Results-In 23 paired myocardial samples, cardiomyocyte ploidy was investigated by DNA image cytometry, flow cytometry, and in situ hybridization. Nuclear cross-sectional area and perimeters were measured morphometrically, and the binucleated cardiomyocytes were counted. The median of the cardiomyocyte DNA content and the number of polyploid cardiomyocytes both declined significantly from 6.79 c to 4.7 c and 40.2% to 23%, whereas a significant increase in diploid cardiomyocytes from 33.4% to 50.3% and in binucleated cardiomyocytes from 4.5% to 10% after unloading was observed. Conclusions-The decrease in polyploidy and increase in diploidy after left ventricular assist device suggest a numeric increase in diploid cardiomyocytes (eg, through cell cycle progression with completion of mitosis or by increased stem cells). The cardiac regeneration that follows may serve as a morphological correlate of the recovery observed in some patients after unloading. (Circulation. 2010;121:989-996.)

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“…This combined dissociation and purification protocol produced a higher quality cell preparation than the enzymatic digestion and ammonium chloride red cell lysis steps commonly reported in the literature for the dissociation of tumoral or normal mammary and other tissues. 17,20 This optimized protocol produced high-quality cellular material in o3 h, and allowed subsequent cell analyses to be performed on the same day.…”

Section: Discussionmentioning

confidence: 99%

“…However, total cell recovery is critical for obtaining sufficient material for experimentation and for assessing the phenotypes of sparse cell subtypes. 17 As viability and integrity are major concerns, the level of apoptosis and necrosis of the dissociated cells must be carefully assessed, especially when cells are to be further used for ex vivo culture, injection into animals or molecular analyses. Poor or selective tissue dissociation can alter the composition of cell subpopulations, thereby biasing analyses and inferred results.…”

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confidence: 99%

Optimization of tumor xenograft dissociation for the profiling of cell surface markers and nutrient transporters

Petit

Massonnet

Maciorowski

et al. 2013

Laboratory Investigation

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Metabolic adaptations and changes in the expression of nutrient transporters are known to accompany tumorigenic processes. Nevertheless, in the context of solid tumors, studies of metabolism are hindered by a paucity of tools allowing the identification of cell surface transporters on individual cells. Here, we developed a method for the dissociation of human breast cancer tumor xenografts combined with quantification of cell surface markers, including metabolite transporters. The expression profiles of four relevant nutrient transporters for cancer cells' metabolism, Glut1, ASCT2, PiT1 and PiT2 (participating to glucose, glutamine and inorganic phosphate, respectively), as detected by new retroviral envelope glycoprotein-derived ligands, were distinctive of each tumor, unveiling underlying differences in metabolic pathways. Our tumor dissociation procedure and nutrient transporter profiling technology provides opportunities for future basic research, clinical diagnosis, prognosis and evaluation of therapeutic responses, as well as for drug discovery and development. Markers of tumor cell metabolism are of valuable importance for basic and clinical cancer research. The identification of metabolic alterations that accompany cancer processes is becoming critical for refining the clinical evaluation and treatment of patients, 1,2 as well as for further drug discovery and development. While metabolomics relies mainly on the quantification of anabolic and catabolic end products, 3 the bidirectional fluxes of nutrients and metabolites, as well as the efflux of toxins and pharmaceutical compounds, are ensured by nutrient transporters. 4 However, the monitoring of metabolite and nutrient transporters at the cell surface has been hampered by the paucity, if not total lack, of exofacial antibodies to such transporters. 5,6 Cell entry of gamma-and deltaretroviruses occurs via recognition of membrane metabolite transporters by retroviral envelope glycoproteins (Env). 7,8 Viral entry occurs following the specific binding of the amino-terminal receptor binding domain (RBD) of an individual Env with its cognate receptor. We designed RBD probes, derived from gamma-and deltaretroviral Env, as specific ligands that bind to these cell surface transporters. Binding of defined ligands allows the quantification of distinct sets of metabolite transporters at the cell surface. 9-13 Furthermore, while cell surface labeling is readily achieved on cultured cell lines and circulating hematopoietic cells, singlecell labeling of solid tumors has remained problematic. Indeed, such analyses require dissociation methods that yield

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Use of a mechanical dissociation device to improve standardization of flow cytometric cytokeratin DNA measurements of colon carcinomas

Cited by 25 publications

References 33 publications

In vitro sensitivity testing of minimally passaged and uncultured gliomas with TRAIL and/or chemotherapy drugs

In vitro sensitivity testing of minimally passaged and uncultured gliomas with TRAIL and/or chemotherapy drugs

Hemodynamic Support by Left Ventricular Assist Devices Reduces Cardiomyocyte DNA Content in the Failing Human Heart

Optimization of tumor xenograft dissociation for the profiling of cell surface markers and nutrient transporters

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