Nucleic Acid Profile of the Emt6 Cell Cycle <i>In Vitro</i>

Watson, James V.; Chambers, S H

doi:10.1111/j.1365-2184.1978.tb00813.x

Cited by 6 publications

(2 citation statements)

References 9 publications

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“…This metachromatic property of acridine orange has been used extensively to study the RNA and DNA content of populations of single cells by flow cytometry in many cell systems (4,5,11,14,15). It has also been shown that denatured single-stranded DNA emits red fluorescence in proportion to the degree of DNA denaturation (6).…”

Section: Discussionmentioning

confidence: 99%

Flow cytometric fluorescence emission spectrum analysis of hoechst‐33342‐stained DNA in chicken thymocytes

et al. 1985

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Hoechst-33342-stained chicken thymocytes were analysed simultaneously on two fluorescence wavelength bands (green and violet) in our custom-built flow cytometer, and two major subsets were identified. In one subset (33% of the total) the emission spectrum remained constant with time, with little change in the respective green and violet fluorescence intensities. In the other subset (42% of the total) the green fluorescence increased during staining, resulting in a considerable change in the green-to-violet ratio, due to a change in the "shape" of the fluorescence emission with time. The data indicate that two binding sites, or two types of binding at the same site, exist in DNA for this dye and that these have different binding energies and, consequently, different fluorescence emission properties. Key terms: Hoechst 33342, DNA binding, fluorescence spectrumDuring a series of experiments in which we used Hoechst-33342 (Ho-33342) as a vital DNA stain for mouse bone marrow, we encountered a number of anomalies. These were not only dependent on the dyeDNA concentration ratio and staining time (which were to be expected) but also on the wavelength at which the emission spectrum was analysed. In an attempt to produce an internal biological standard, we repeated the staining procedures with chicken thymocytes (CTHY) and discovered similar anomalies in the "DNA" histograms.The Ho-33342DNA emission spectrum is wide and covers a range from about 400 to 600 nm. Our custombuilt flow cytometer, for which preliminary descriptions have been published (12,13) has the capacity to measure fluorescence emission for up to five different wavelength bands simultaneously. During these experiments we noted that the "DNA" histograms obtained with Ho-33342 staining were considerably different when measured on the violet (390-440 nm) and the green (515-560 nm) photomultipliers. This paper describes our experiences with the chicken thymocytes and discusses some possibilities for the phenomena observed.phate-buf€ered saline, PBS (pH 7.21, the cells were resuspended in Iscove's serum-free medium, pH 7.2, at a concentration of 106/ml. StainingA stock solution of Hoechst-33342 (Calbiochem, La Jolla, CA) was dissolved in distilled water at a concentration of 1 mM. Aliquots of cells were then stained by adding either 1 or 5 p1 of the stock Ho-33342 solution to 1 ml of the cell suspensions to give final stain concentrations of either 1 or 5 pM. The stained cells were then incubated at 37°C in the dark. The samples were analysed at 15, 30, 45, and 125 min after addition of the stain. Flow CytometryThe 164-05 argon laser (Spectra-Physics, Mountainview, CA) of our flow cytometer was tuned to the UV lines (351 + 363 nm), and the fluorescence emission spectrum was analysed on two photomultiplier tubes that admitted light through bandpass filters of 390-440 MATERIALS AND METHODS CellsThymocytes were obtained from a 6-wk-old commercial white-leghorn-based hybrid chicken. A single cell suspension was obtained by teasing the thymus and filtering through ...

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Section: Discussionmentioning

confidence: 99%

Flow cytometric fluorescence emission spectrum analysis of hoechst‐33342‐stained DNA in chicken thymocytes

et al. 1985

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“…In our study, the cells in fractions 1 and 2 lie within the growth fraction whilst the cells in the R fraction are from the inner region of the viable rim. Cells in fraction 3 are from the transitional zone between them, Rapidly dividing EMT6 monolayer cells were reported to have higher cellular RNA content than non-dividing cells (Watson and Chambers, 1978). It is therefore reasonable to suppose that the RNA content of cells in the outer region of the spheroids is higher than that of cells in the inner region.…”

Section: Characteristics Of Cells Ffom Different Fractions Of Spheroidmentioning

confidence: 99%

The relationship between tumour geometry and the response of tumour cells to cytotoxic drugs — An in vitro study using EMT6 multicellular spheroids

Kwok

Twentyman

1985

Intl Journal of Cancer

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Multicellular spheroids of the EMT6/Ca/VJAC mouse mammary tumour cell line have been used in an investigation of the effect of tumour geometry on the response of tumour cells to 3 cytotoxic drugs, adriamycin (ADM), nitrogen mustard (HN2) and CCNU. In addition to the inherent cellular drug response, factors related to spheroid structure, namely cell-cycle distribution, intercellular contact, drug penetration and microenvironment (pH, oxygen, glucose, etc.) are believed to influence the response of cells within spheroids to cytotoxic drugs. Selective enzymatic dissociation (with bacterial neutral protease) has been used to separate the cells within large (approximately 800 micron in diameter) spheroids into 4 distinct subpopulations. The cells within the subpopulations have been characterized by their DNA content, RNA content, tritiated thymidine labelling index, cell size and clonogenic capacity. It was found that cells at the surface of spheroids are relatively larger and more proliferative than cells towards the centre while their clonogenic capacity is similar. Studies on the responses of EMT6/Ca/VJAC log and plateau-phase monolayer cells have been carried out in parallel and have shown that cycling cells are more sensitive to ADM and HN2 than are non-cycling cells but somewhat less sensitive for the response to CCNU. Since the response patterns of cells from different regions of spheroids to HN2, treated either before disaggregation (intact spheroid) or after disaggregation (isolated spheroid cells), are similar and the surviving fraction increases from the surface towards the centre of the spheroid, cell cycle distribution is thought to be the only factor involved in the cytotoxicity of HN2 towards cells within the spheroids. Although the patterns of response to ADM of cells within intact spheroids and isolated spheroid cells are similar to those for HN2, the initial slope of the curve for intact spheroids is much steeper than that of the isolated spheroid cells. Therefore, in addition to the factor of cell-cycle distribution, drug penetration also appears to be involved in the action of ADM on spheroids, while the factors of intercellular contact and microenvironment appear to be relatively less important. The reverse pattern was found for the response of cells within different regions of spheroids to CCNU, treated as intact spheroids or as isolated spheroid cells (i.e., greater killing of inner compared with outer cells).(ABSTRACT TRUNCATED AT 400 WORDS)

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Molecular interactions and cellular changes during the cell cycle

Darzynkiewicz

1983

Pharmacology & Therapeutics

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Nucleic Acid Profile of the Emt6 Cell Cycle In Vitro

Cited by 6 publications

References 9 publications

Flow cytometric fluorescence emission spectrum analysis of hoechst‐33342‐stained DNA in chicken thymocytes

Flow cytometric fluorescence emission spectrum analysis of hoechst‐33342‐stained DNA in chicken thymocytes

The relationship between tumour geometry and the response of tumour cells to cytotoxic drugs — An in vitro study using EMT6 multicellular spheroids

Molecular interactions and cellular changes during the cell cycle

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