Summary
This report describes the results of the comparison of three different methods and three monoclonal antibodies to stain cells in suspension for incorporated bromodeoxyuridine and total DNA content. The procedures were tested in three different experimental tumour cell lines. The sensitivity of the different procedures was expressed as the ratio of the anti-BrdUrd fluorescence intensities of the S and G1 phase cells (FS/FG1 ratio). There were remarkable differences in sensitivity between the different procedures. With the heat denaturation the most favourable FS/FG1 ratio's were obtained but substantial cell loss occurred during this procedure which is a disadvantage for clinical application. With the pepsin digestion + acid denaturation procedure cell loss was negligible. The standard acid denaturation procedure was inferior to the other two methods. Using the pepsin digestion + acid denaturation procedure we examined the variations in sensitivity for the different monoclonal antibodies and cell lines and the influence of BrdUrd concentration, labelingtime and cell concentration. The binding characteristics for the various antibodies differed considerably in our hands. Only with the IU4 antibody we obtained FS/FG1 ratio's comparable with those desenbed in the literature. No difference was observed between the cell lines. Variation in cell concentration between 1 × 104 to 1 × 106 ml nor BrdUrd concentration appeared to influence the sensitivity of the procedure. A labelingtime of 1 h or even 30 min seems to be more than sufficient for an optimal FS/FG1 ratio.
Our results indicate that using the appropriate antibody and immunofluorescence BrdUrd can be detected by flow cytometry, after incorporation into the DNA of tumour cells under a wide range of culture conditions.
For clinical application, the pepsin digestion + acid dena uration method in combination with IU4 antibody seems to be the procedure of choice due to its good reproducibility, sensitivity and its low cell loss.
Similar content being viewed by others
References
Barendsen GW, Jansen HC, Deys BF, Hollander CF (1977) Comparison of growth characteristics of experimental tumours and derived cell cultures. Cell Tissue Kinet 10:469–475
Begg AC, McNally NJ, Schrieve DC, Karcher H (1985) A method to measure the duration of DNA synthesis and the potential doubling time from a single sample. Cytometry 6:620–626
Beisker W, Dolbeare, F, Gray JW (1987) An improved immunocytochemical procedure for high-sensitivity detection of incorporated bromodeoxyuridine. Cytometry 8:235–239
Dolbeare F, Gratzner H, Pallavicini MG, Gray JW (1983) Flow cytometry measurement of total DNA content and incorporated bromodeoxyuridine. Proc Natl Acad Sci USA 80:5573–5577
Dolbeare F, Beisker W, Pallavicini MG, Vanderlaan M, Gray JW (1985) Cytochemistry for bromodeoxyuridine/DNA analysis: Stoichiometry and sensitivity. Cytometry 6:521–530
Gratzner H (1982) Mohoclonal antibody against 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication. Science 218:474–475
Schutte B, Reynders MMJ, van Assche CLMVJ, Hupperets PSGJ, Bosman FT, Blijham GH (1987) An improved method for the immunocytochemical detection of bromodeoxyuridine labeled nuclei using flow cytometry. Cytometry 8:372–376
Vanderlaan M, Watkins B, Thomas C, Dolbeare F, Stanker L (1986) Improved high-affinity monoclonal antibody to iododeoxyuridine. Cytometry 7:499–507
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Bakker, P.J.M., Aten, J.A., Tukker, C.J. et al. Flow cytometric analysis of experimental parameters for the immunofluorescent labeling of BrdUrd in various tumour cell lines. Histochemistry 91, 425–429 (1989). https://doi.org/10.1007/BF00493830
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00493830