Improved single laser measurement of two cellular antigens and DNA-ploidy by the combined use of propidium iodide and TO-PRO-3 iodide

Corver, Willem E.; Fleuren, Gert Jan; Cornelisse, Cees J.

doi:10.1002/(sici)1097-0320(19970801)28:4<329::aid-cyto9>3.0.co;2-6

Cited by 12 publications

(7 citation statements)

References 13 publications

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“…The approach we present here can be considered a follow‐up study. It extends previous developments of multiparametric approaches (27–29) in which the potential was indicated to account for tumor heterogeneity in a sample, through flow cytometric phenotyping and DNA quantification (25, 30–35).…”

Section: Discussionsupporting

confidence: 57%

Multichromatic phenotyping of HER receptor coexpression in breast tumor tissue samples using flow cytometry—Possibilities and limitations

et al. 2010

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The prognostic significance of HER2 expression in human breast carcinomas is beyond dispute nowadays. The HER family of receptor tyrosine kinases comprises four members (HER1/ErbB1/EGFR, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4) that act in concert via transactivation and consequently compose a functional signaling unit. Besides HER2 overexpression, coexpression of other HER receptors has substantial impact on course of disease and potential therapeutic benefit. This observation is substantiated by numerous preclinical studies and retrospective studies done on patients with breast cancer. Against this background, the quantification of all HER receptor expressions at the same time would significantly extend the information content revealed by routine diagnosis of breast cancer tissues. Moreover, the knowledge of HER receptor coexpression profiles in primary tumor samples could provide the basis to design and develop highly specific antireceptor treatment strategies. Here, we report on a simultaneous flow cytometric detection of all four HER receptors on carcinoma cells isolated from primary breast cancer tissues and separated from nonepithelial cells by cytokeratin staining. Combined with DNA, i.e. ploidy quantification, the approach resulted in a six-parameter assay that could complement the diagnosis of a variety of diseases in which HER receptor expression has a pivotal impact on the degree of malignancy. ' 2010 International Society for Advancement of Cytometry Key terms receptor tyrosine kinases; multiparametric flow cytometry; solid tumor dissociation; DNA assessment THE oncogenic role of EGFR and HER2 tyrosine kinases in a variety of epithelial tumors has led to the development of a number of targeted anticancer drugs, for example the therapeutic antibody Trastuzumab (directed against HER2) and the small tyrosine kinase inhibitors (TKIs) ZD 1839 (Gefitinib, directed against EGFR) and Lapatinib (targeting both receptors EGFR and HER2). Although those agents evidently affect or inhibit individual HER receptors both in vitro and in vivo, the response rate in patients remains poor. These findings strongly indicate that diagnostic approaches addressing the expression density of a single type HER receptor do not predict drug responsiveness. However, the molecular characteristics of breast cancer cells contributing to target specific susceptibility resistances have not been fully described.The HER family of receptor tyrosine kinases (RTKs) comprises two additional members, namely HER3 and HER4. Their function in breast cancer development and progression has been less extensively explored; however, there is a growing body of evidence suggesting that crosstalk between the four different receptors may play a crucial role in mediating therapy responsiveness (1,2). All receptors share a high degree of homology and are able to interact with and cross-activate each other. Different homo-and heterodimers can be formed and the availability of dimerization partners as well as of activating ligands defines the phospho...

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Section: Discussionsupporting

confidence: 57%

Multichromatic phenotyping of HER receptor coexpression in breast tumor tissue samples using flow cytometry—Possibilities and limitations

et al. 2010

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“…Then, a stock of TO‐PRO‐3 iodide of 1 × 10 −3 M (TP3, Molecular Probes, Eugene, OR) was diluted 500 times and 300 μl of this solution was then added to each sample, resulting in a final concentration of 2 × 10 −5 M. TP3 gives good results with no spectral overlap with phycoerythrin. Moreover, compared with propidium iodide, often considered the standard for DNA staining, TP3 shows similar results (8, 43, 44). TP3 intercalates with double‐stranded regions of nucleic acids, but it also affects RNA.…”

Section: Methodsmentioning

confidence: 82%

Novel functional multiparameter flow cytometric assay to characterize proliferation in skin

2000

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Keratins are a group of cytoskeletal proteins that are found in human epidermis and other stratified squamous epithelia. Several different types of keratins have been described. Keratin 10 (K10) is a keratin that is expressed in well differentiated, suprabasal keratinocytes, and keratin 6 (K6) is a keratin which is associated with hyperproliferation. Psoriasis is a chronic inflammatory skin disease, and besides inflammation, disturbed differentiation and hyperproliferation are its hallmarks. In order to study the hyperproliferation associated keratinization in both well differentiated and poorly differentiated keratinocytes, and in order to assess the proliferative activity of all K10 and K6 subpopulations, simultaneous assessment of K6, K10, and DNA content is required. So far, a triple staining protocol had not been available. In the present study, we established a novel protocol for simultaneous measurement of K6, K10, and DNA content, which enables the characterization of the proliferative activity of several cellular subpopulations in epidermis. From 16 patients with psoriasis and from 15 healthy volunteers, punch biopsies were obtained. After preparation of single cell suspensions, cells were stained with the anti‐keratin 10 IgG1‐isotype monoclonal antibody RKSE60, with the anti‐keratin 6 IgG2a‐isotype monoclonal antibody LHK6B, and with the DNA fluorochrome TO‐PRO‐3 iodide. Isotype specific secondary antibodies conjugated with phycoerythrein (PE) and fluorescein isothiocyanate (FITC) were used as the second step in the staining procedure. Controls were measured omitting the primary antibodies, and gates were set in order to differentiate between the K10 and K6 subpopulations. Samples from both psoriatic patients and healthy volunteers were than measured. Owing to the IgG specificity of RKSE60 and LHK6B, no cross‐reactivity was observed between these antibodies. The triple staining with RKSE60, LHK6B, and TO‐PRO‐3 iodide showed subpopulations of K10 expressing cells, K10/K6 co‐expressing cells, and K6 only expressing cells. There was a significant difference in the proportion of K6 expression and K10/K6 co‐expression between psoriatic and normal skin. Moreover, the proliferative activity of these subpopulations could be quantified by this protocol. We concluded that a triple staining protocol for the assessment of K6, K10, and DNA content, using the monoclonal antibodies LHK6B, RKSE60, and TO‐PRO‐3 iodide, supplies reliable and reproducible data for cellular studies on these keratins and for studying the proliferative activity of the subpopulations of these keratins in epidermis. Moreover, the present study showed that with respect to the proportion of K6, significant differences are present between psoriatic and healthy human skin.Cytometry (Comm. Clin. Cytometry) 1:43–49, 2000 © 2000 Wiley‐Liss, Inc.

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“…The use of anti‐keratin monoclonal antibodies (mAbs) enables the identification of carcinoma cells in cell suspensions from solid tumors. Simultaneously, these cells can be stained for a second cellular protein, e.g., p53 (6, 7), the epithelial cell adhesion molecule (Ep‐CAM), erbB2 (2), or epidermal growth factor receptor (EGFR) (3), and DNA. Fluorescein‐isothiocyanate (FITC) and R‐phycoerythrin (RPE) are frequently used as fluorescent reporter molecules for cellular proteins, and DNA is stained with propidium iodide (PI).…”

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confidence: 99%

Four-color multiparameter DNA flow cytometric method to study phenotypic intratumor heterogeneity in cervical cancer

Corver

Koopman

Aa³

et al. 2000

Cytometry

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BACKGROUND Multiparameter DNA flow cytometry using a one‐laser bench‐top flow cytometer has been restricted to three different colors. The two laser FACSCalibur has recently been introduced, allowing four‐color analysis. Therefore, we optimized and extended our three‐color method (Corver et al., 1994, Corver et al. 1996) to a four‐color analysis of phenotypic intra‐tumor heterogeneity using a bench‐top flow cytometer. METHODS First, the effect of a range of different propidium iodide (PI) and TO‐PRO‐3 iodide (TP3) concentrations on the coefficient of variation (CV) of the DNA histograms was measured using paraformaldehyde‐fixed lysolecithin‐permeabilized peripheral blood lymphocytes (PBLs) and SiHa and HeLa cervical cancer cells. Second, labeling freshly isolated cervical cancers from solid tumors was optimized with a mixture of anti‐keratin antibodies. Third, the FACSCalibur hardware was modified, thereby allowing the simultaneous measurement of allophycocyanin (APC) fluorescence (FL4) in combination with FL3 pulse processing (FL3‐W vs. FL3‐A). The optimized procedure was then applied to cell suspensions from four different human cervical cancers to study phenotypic intratumor heterogeneity. Cell suspensions were simultaneously stained for DNA (PI, fluorescence) and three cellular antigens: (a) the epithelial cell‐adhesion molecule (Ep‐CAM; APC fluorescence), (b) keratin (R‐phycoerythrin [RPE] fluorescence) to identify the epithelial fraction, and (c) vimentin (fluorescein‐isothiocyanate [FITC] fluorescence) to label stromal cells. RESULTS Overall, PI produced better CVs than did TP3. The optimal concentration of PI was 50–100 μM for all cells tested. Average CVs were 1.76% (PBL), 3.16% (HeLa), and 2.50% (SiHa). Optimal TP3 concentrations were 0.25–2.0 μM. Average CVs were 2.58% (PBL), 5.16% (HeLa), and 3.96% (SiHa). Inter‐ or intra‐DNA stem line heterogeneity of Ep‐CAM expression was observed in the keratin‐positive fractions. Vimentin‐positive, keratin‐negative cells were restricted to the DNA diploid fraction. CONCLUSIONS PI is a superior DNA stain to TP3 when using intact normal PBL and human cancer cells. Four‐color high‐resolution multiparameter DNA flow cytometry allows the identification of intratumor subpopulations using PI as DNA stain and FITC, RPE, and APC as reporter molecules. The FACSCalibur bench‐top flow cytometer can be used for this purpose, allowing the application of this technique in clinical laboratories. Cytometry 39:96–107, 2000 © 2000 Wiley‐Liss, Inc.

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Improved single laser measurement of two cellular antigens and DNA-ploidy by the combined use of propidium iodide and TO-PRO-3 iodide

Cited by 12 publications

References 13 publications

Multichromatic phenotyping of HER receptor coexpression in breast tumor tissue samples using flow cytometry—Possibilities and limitations

Multichromatic phenotyping of HER receptor coexpression in breast tumor tissue samples using flow cytometry—Possibilities and limitations

Novel functional multiparameter flow cytometric assay to characterize proliferation in skin

Four-color multiparameter DNA flow cytometric method to study phenotypic intratumor heterogeneity in cervical cancer

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