| 1. |
- Johansson, Maria C, et al.
(författare)
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Comparison of different labelling index formulae used on bromodeoxyuridine-flow cytometry data
- 1998
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Ingår i: Cytometry. - 0196-4763. ; 32:3, s. 233-240
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Tidskriftsartikel (refereegranskat)abstract
- The essence of the bromodeoxyuridine (BrdUrd)-flow cytometry (FCM) technique is that cells are labelled with the thymidine analogue BrdUrd. They are then allowed to progress through the cell cycle in a BrdUrd-free environment during the postlabelling time period. At a postlabelling time shorter than the length of the S phase (Ts), cells are fixed and prepared for FCM-mediated analysis of BrdUrd and DNA contents. From FCM-derived data, cell cycle kinetic parameters such as labelling index (LI), Ts, and potential doubling time (Tpot) can be calculated. Tpot is believed to be important in the evaluation of tumor aggressiveness and therapy response. Since LI is most commonly used together with Ts to calculate Tpot, it is important that both LI and Ts are independent of the time when cells are sampled. Several formulae to calculate LI and Ts have been presented. In the present paper, we deal with various formulae to calculate LI. These formulae differ in how they take into account unlabelled and BrdUrd-labelled cells in various fractions of the cell cycle. We present a new formula, which takes into consideration cells in the different fractions and thus makes LI theoretically independent of postlabelling time. Our results show that different LI values are obtained when different formulae are used to calculate LI. In addition, we show that the BrdUrd labelling period should be kept as short as possible.
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| 2. |
- Johansson, Maria C, et al.
(författare)
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Comparison of mathematical formulas used for estimation of DNA synthesis time of bromodeoxyuridine-labelled cell populations with different proliferative characteristics
- 1996
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Ingår i: Cell Proliferation. - : Wiley. - 1365-2184 .- 0960-7722. ; 29:10, s. 525-538
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Tidskriftsartikel (refereegranskat)abstract
- Growth kinetic data of human tumours, obtained by flow cytometric analysis of cells labelled with bromodeoxyuridine (BrdUrd) might provide prognostic information and allow prediction of response to radio- and chemotherapy. However, the theoretical models applied for calculation of growth kinetic data are not fully evaluated. The purpose of this study was to investigate the dependence of the estimation of DNA synthesis time (Ts) on sampling time after BrdUrd labelling, using four different mathematical formulas (Begg et al., White & Meistrich, White et al. and Johansson et al.) which have been developed for the evaluation of flow cytometry-derived data of BrdUrd-labelled cells. In addition, we have investigated the influence of the growth kinetic properties of the cell populations using two cultured cell lines (one slow and one fast growing), and two hetero-transplanted human tumours. The dependence of the estimation of Ts on sampling time was more or less pronounced, depending on the cell population examined and on the formula used. In the fast growing cell line, the estimates of Ts did not vary significantly with sampling time when using the formulas by White et al., whereas in the slow growing cell line, the estimates of Ts did not show any significant dependence on sampling time when using the formula by Johansson et al. In the tumours, the estimation of Ts depended on sampling time with all formulas used, although to different degrees. In one of the tumours, this was mainly caused by the influence of mouse cells, as we demonstrate. Our results indicate that the proliferative characteristics of a cell population should be taken into consideration when choosing a mathematical formula in order to attain Ts values that are independent of sampling time.
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| 3. |
- Johansson, Maria C, et al.
(författare)
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Energy transfer between fluorescein isothiocyanate and propidium iodide--a problem in the estimation of Tpot with the bromodeoxyuridine-DNA flow cytometry technique?
- 1999
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Ingår i: Analytical Cellular Pathology. - 0921-8912. ; 19:2, s. 91-98
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Tidskriftsartikel (refereegranskat)abstract
- Energy transfer in flow cytometry can occur when two fluorochromes are bound in close proximity (generally within 100 A) and the emission spectrum of one fluorochrome overlaps significantly with the excitation spectrum of the other. The latter criterium is fulfilled for the fluorochromes fluorescein isothiocyanate and propidium iodide and also the former when they, e.g., are used in bromodeoxyuridine - DNA flow cytometry methods. In the present growth kinetic study using this method, we show that energy transfer does take place between fluorescein isothiocyanate and propidium iodide which results in a detected increase in DNA content with 2-3%. Despite the erroneous increase in the obtained DNA content values, this does not seem to have any influence on the calculation of DNA synthesis time and potential doubling time where the DNA content, based on the relative movement principle of the labelled cells, is used.
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