Cytometry 12:366-372 (1991)

0 1991 Wiley-Liss, Inc.

An Indirect Immunof luorescence Double Staining Procedure for the Simultaneous Flow Cytometric Measurement of Iodo- and Chlorodeoxyuridine Incorporated into DNA P.J.M. Bakker,' J. Stap, C.J. Tukker, C.H. van Oven, C.H.N. Veenhof, and J. Aten Division of Medical Oncology (P.J.M.B., C.H.N.V.),Department of Medicine, and Laboratory for Radiobiology (J.S.,C.J.T., C.H.V.O., J.A.), Academic Medical Center, Amsterdam, the Netherlands Received for publication May 23, 1990; accepted December 18, 1990

In this paper we describe an indirect fluorescence double staining procedure for the simultaneous detection of IdUrd and CldUrd in the same cell nucleus. Two commercially available antibodies were selected for this purpose. A rat antiBrdUrd monoclonal antibody from Seralab was found to bind specifically to CldUrd and BrdUrd. A mouse monoclonal anti-BrdUrd antibody from Becton Dickinson used in a 1:2 dilution binds to all halogenated deoxyuridines but, when the cells were extensively washed with

The study of experimental and human tumors has been facilitated by the recent introduction of immunocytochemical procedures for the detection of halogenated deoxyuridines incorporated into cellular DNA (1,2,4-11,13,15,17,18). These methods are rapid and easy to perform, and data from a single sample can provide several cell kinetic parameters (cf. cell cycle time, duration of S-phase, and the potential doubling time) ( 3 ) . However, not all relevant cell cycle kinetic parameters can be studied by this technique. Recruitment of cells can only be studied when a combination of DNA labels is used that can be detected separately. Detection of a second label in the same cell population requires the development of a n immunocytochemical double staining procedure using a pair of monoclonal antibodies with high specificity for different halogenated deoxyuridines and with low cross-reactivity. Most commercially available antibodies show crossreactivity between the two most frequently used halogenated deoxyuridines: bromo- and iododeoxyuridine (BrdUrd and IdUrd). Monoclonal antibodies with different specificities were reported by Vanderlaan et al. (16): Br-3, which should only recognize BrdUrd, and IU-4, which recognizes both BrdUrd and IdUrd. Shibui et al. (14) recently developed a n immunocytochemical

Tris buffer with a high salt concentration, almost no binding to CldUrd was observed. An immunofluorescence procedure was developed, based on these primary antibodies, raised in different species (rat and mouse), in combination with highly purified second antibodies: FITC conjugated goat antirat and TexasRed conjugated goat antimouse. Key terms: Immunochemistry, double staining procedure, IdUrd, CldUrd, flow cytometry, cell kinetics

staining procedure using these antibodies for the detection of IdUrd and BrdUrd given to the same cell population. A major disadvantage of this procedure is that the IU-4 antibody recognizes both labels IdUrd and BrdUrd. Moreover, since this reaction is based on enzyme reactions, it is not suitable for cells in suspension, hence is unsuitable for flow cytometric application. We recently studied several commercially available monoclonal antibodies for their specificity for binding to bromo-, iodo-, and chlorodeoxyuridine. Although nearly all monoclonal antibodies examined reacted with all the different halogenated deoxyuridines, we were able to select a pair of antibodies that, under the chosen experimental conditions, showed a large difference in binding to IdUrd and CldUrd. Using these commercially available antibodies, we were able to develop a n indirect fluorescence double staining procedure for the simultaneous detection of IdUrd and CldUrd, suitable for FCM analysis.

'Address reprint requests to Dr. P.J.M. Bakker, Division of Medical Oncology, F4-224, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands.

DOUBLE STAINING FOR SIMULTANEOUS FCM OF IdUrd AND CldUrd INTO DNA

MATERIALS AND METHODS Cell Culture Cultures from a V79 Chinese hamster and a mouse osteosarcoma (MOS) cell line were grown a s monolayers in Costar tissue culture flasks. The cultures were incubated in Eagle’s minimum essential medium with Hank’s balanced salt solutions, supplemented with 10% fetal calf serum, glutamin, and penicillin in a n atmosphere of 2% COz in air a t 37°C.

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antibodies are purified by immunoaffinity chromatography to remove antibodies t h a t cross-react with human, bovine, horse, and mouse serum proteins.

Single Labeling and Staining Experiments A number of experiments were performed to test the specificity of the two monoclonal antibodies selected. Cells labeled with either bromo-, iodo-, or chlorodeoxyuridine were partially denaturated and then resuspended in 100 p1 PBT buffer containing monoclonal anti-BrdUrd antibody (Becton Dickinson) diluted 1:2 or Labeling Procedure in 100 pl PBT buffer containing the monoclonal antiFor all experiments, cells from exponentially grow- BrdUrd antibody (Sera-lab)diluted 1:600. After 30 min ing cultures were used. In the single labeling experi- a t room temperature, the cells were sedimented, resusments, cells were pulse labeled by incubation with bro- pended, and incubated for 30 min in 5 ml Tris buffer modeoxyuridine (BrdUrd), iododeoxyuridine (IdUrd), containing 29.22 g NaCl/L, 4.44g Tris HClIL, 2.65 g or chlorodeoxyuridine (CldUrd) containing medium Tris base/L, and 0.5% Tween-20 (pH 8.0). Cells were (final concentration 10 pM) for 30 min. In the double then sedimented and resuspended in 1ml PBT containlabeling experiments, cells were incubated for 30 min. ing normal goat serum 1mg/ml (Dakopatts, no. X 907). with iododeoxyuridine, after which the medium was Subsequently the cells were sedimented, resuspended, removed. The cells were then washed three times with and incubated for 30 min a t room temperature in 100 prewarmed normal medium. After culturing in normal pl fluorescein conjugated goat antimouse IgG (Tago, medium for 3 h, the cells were pulse labeled with chlo- no. 6250) or in 100 p1 fluorescein conjugated goat anrodeoxyuridine for 30 min, after which they were har- tirat IgG (Jackson, no. 112-015-102) both diluted 1:lOO vested. in PBT supplemented with goat serum 1 mgiml. The cells were then washed twice in PBT and finally susImmunocytochemical Staining Procedures pended in 1 ml PBS containing 10 pg/ml propidium In this protocol, all centrifugations were carried out iodide (PI). The sensitivity of the labeling was exfor 1 min a t 500g using a Hettich Rotixa/KS type 7250 pressed as the ratio of the mean green fluorescence (F) centrifuge. The cells were harvested by trypsinization, of the cells in the S-phase region of DNA content t o the centrifuged, and resuspended in 2 ml phosphate-buff- mean green fluorescence of the cells in the G1-phase ered saline (PBS) and fixed by adding 6 ml cold ethanol region of DNA content (FSIFGl ratio). (final concentration +70%) for at least 30 min. Fixed To test the specificities of the secondary antibodies, cells could be stored at 4°C for 1 3 weeks. Approxi- the same procedure was used. IdUrd-labeled cells were mately 1 x lo6 ethanol-fixed cells were centrifuged, incubated with the monoclonal anti-BrdUrd antibody resuspended, and incubated in 1 ml pepsin solution (Becton Dickinson) but instead of Texas-Red conju(Boehringer Mannheim GmbH, FRG, 108057, 0.4 mg/ gated goat antimouse IgG, fluorescein conjugated goat ml in 0.1 M HC1) for 30 min at room temperature. The antirat IgG was added. CldUrd labeled cells were inenzymatic treatment was terminated by adding 3 ml cubated with the monoclonal anti-BrdUrd antibody PBS buffer. The cells were then centrifuged, partial (Sera-lab) and subsequently incubated with Texas-Red denaturation was carried out by adding 1 m12 M HC1 conjugated goat antimouse IgG. In this case, the profor 30 min at 37°C (15). After adding 3 ml borax buffer pidium iodide was replaced by DAPI in a final concen(0.1 M sodium tetraborate, pH 8.5) to neutralize the tration of 10 pg/ml. HC1, cells were sedimented and resuspended in 1 ml Double Labeling and Double PBT (PBS, Tween-20 0.05% v/v, pH 7.4) supplemented Staining Experiments with bovine serum albumin (BSA) 1mg/ml. Cells could then be stained for bromodeoxyuridine, iododeoxyuriIn the double staining experiments, cells were either dine, and/or chlorodeoxyuridine. individually labeled with IdUrd or CldUrd and then The two monoclonal antibodies selected for the mixed or double labeled first with iododeoxyuridine double staining procedure were the rat anti-BrdUrd and 3 h r later with chlorodeoxyuridine. Subsequently, (Sera-lab, MAS 250 C, clone Bu/75) and the mouse anti- the cells were partially denaturated, sedimented, and BrdUrd (Becton Dickinson, no. 7580). Other commer- resuspended in 100 pl PBT buffer containing monocially available monoclonal antibodies tested showed clonal anti-BrdUrd antibody (Becton Dickinson) ditoo much cross-reactivity. An indirect immunof luo- luted 1:2. After 30 min at room temperature, the cells rescent staining procedure was used to detect the ha- were sedimented, resuspended, and incubated for 30 logenated nucleotides. Texas-Red conjugated goat an- min in 5 ml Tris buffer. Cells were then sedimented timouse IgG (Jackson, no. 115-075-100) and the and resuspended in 1 ml PBT containing normal goat fluorescein-conjugated goat antirat IgG (Jackson no. serum 1 mg/ml (Dakopatts, no. X 907). Subsequently, 112-015-102)were used as secondary antibodies. These the cells were sedimented, resuspended, and incubated

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Table 1 Halogenated Deoxyuridines"

Anti-BrdUrd monoclonal antibodies Becton Dickinson (no. 758) Sera-lab (Mas 250c clone Bui75)

Species Mouse

CldUrd l b

(k1.0) 21.8 (213.0)

Rat

FS/FGl ratio BrdUrd 16.9 (k2.1) 38.0 (210.7)

IdUrd 21.3 (29.5) 1 (21.0)

"The binding of the two monoclonal antibodies to different halogenated deoxyuridines expressed as the mean relative fluorescence intensity of S-phase cells and GI-phase cells (FSIFGl ratio). Each value represents the average of three or more independent experiments ( k l x SD). 'This result was obtained after extensive washing, two times 30 min with 5 ml Tris buffer, with a high salt concentration.

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ing of DNA. b. CldUrd labeled cells were stained with the rat antiBrdUrd monoclonal antibody (Sera-lab) and Texas-red conjugated goat antimouse IgG. DAPI was used for the staining of DNA.

for 30 min in 100 p1 PBT containing normal goat serum 1 mgiml (Dakopatts, no. X 907) and Texas-Red conjugated goat antimouse IgG (Jackson, no. 115-075-100). Thereafter, cells were sedimented and resuspended in 1 ml PBT supplemented with BSA 1 g/ml and subsequently sedimented, resuspended, and incubated for 30 min in 100 pl PBT buffer containing the monoclonal anti-BrdUrd antibody (Sera-lab) diluted 1:600. The cells were then sedimented, resuspended, and incubated for 30 min in Tris buffer. Thereafter, cells were sedimented and resuspended in 1 ml PBT containing goat serum 1 mgiml and sedimented, resuspended, and incubated for 30 min a t room temperature in 100 p1 PBT with normal goat serum (1 mg/ml) and fluorescein-conjugated goat antirat IgG (Jackson, no. 112-

1015-102) 1:lOO. Finally, they were sedimented and resuspended in 1ml PBS. To test for the degree of nonspecific binding in the double antibody staining technique, unlabeled cells underwent the same double staining procedure.

DNA Histograms and Cell Loss The effect of the overall technique on the different cell cycle populations was tested by comparing the DNA histograms of unlabeled cells with the DNA histograms of cells that have gone completely through the double labeling and two antibody staining technique. Labeled and unlabeled cells were cultured from the same cell populations and plated in the same numbers. To produce DNA histograms, the unlabeled cells were

369

DOUBLE STAINING FOR SIMULTANEOUS FCM OF IdUrd AND CldUrd INTO DNA 64

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Texas-Red fluorescence

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FIG.2. Bivariate IdUrd/CldUrd distribution from asynchronously growing MOS cells, all stained using the double staining procedure. a. Unlabeled cells. b. Cells labeled with IdUrd. c. Cells labeled with CldUrd. d. Cells individually labeled with either IdUrd or CldUrd and then mixed before staining.

trypsinated, sedimented, and resuspended in 0.25 ml culture medium and mixed with 0.25 ml RNase solution (Sigma, type lA, 4 mg/ml Tris buffer) and 0.5 ml propidium iodide + saponine solution and incubated for 10 min at 37°C. The propidium iodide + saponine solution contained 0.05 mg propidium iodide (Celbiochem, no. 537059)/m1 Tris buffer, 0.2 mg saponine (OPG, Utrecht, the Netherlands)/ml, and lop3 mg Triton X-lOO/ml. Tris buffer contained 0.1 M Tris, 0.1 M NaC1, and 0.005 M EDTA 2 aq (pH 7.5). The percentages of cells in each subcompartment of the cell cycle were derived using a calculation procedure (12). Cell loss was calculated by counting cells before the staining procedure and again before flow cytometric analysis using a haemocytometer. Before flow cytometric analysis, the cells were syringed through a 21 gauge needle t o disaggregate cell clumps. All experiments were carried out at least three times.

Flow Cytometry For the single labeling and staining experiments, we used the Cytofluorograph 50 flow cytometer with a Spectra Physics 2020 argon ion laser tuned at 488 nm.

The fluorescence light was intercepted by a dichroic mirror (590 nm). The FITC and PI fluorescence signals were detected simultaneously through a 520-nm bandpass filter and a 630-nm-long pass filter (Schott RG 6301, respectively. Two Spectra Physics 2020 argon ion lasers tuned to 488 nm and 350 nm, respectively, were used for excitation of the Texas-Red, or DAPI molecules. The cells first passed through the 350-nm beam and 40 ps later, through the 488-nm beam. The fluorescence light was intercepted by a blue-reflecting dichroic mirror (500 nm). The Texas-Red fluorescence signal was detected through a 610-nm-long pass filter. The DAPI fluorescence signal was detected through a 380-nm bandpass filter (Schott BG25). In the double antibody staining experiments cell suspensions were analyzed using a Cytofluorograph 50 flow cytometer with two Spectra Physics 2020 argon ion lasers tuned to 488 nm (200 mW) and 514 nm (600 mW) for excitation of the FITC and Texas-Red molecules, respectively. The cells first passed through the 514 nm beam and 40 p s later through the 488-nm beam.

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514-nm scatter signal. The fluorescence light was separated into green and red fluorescence signals using a dichroic mirror (590 nm, Ortho part no. 390-0105-000) supplied with the cytofluorograph. The FITC and Texas-Red fluorescence signals were detected through a 520-nm bandpass filter and a 630-nm-longpass filter, respectively. The data were accumulated in a two-dimensional array of 64 x 64 channels and displayed as linear bivariate contour plots.

0

64

Texas-Red fluorescence

FIG.3. Bivariate IdUrdCldUrd distribution in a double labeling experiment with asynchronously growing MOS cells using the double staining procedure.

By using the standard dual-beam signal analysis system of the Cytofluorograph, the fluorescence signal of the FITC conjugated cells can be separated from the

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RESULTS The relative specificities of the various antibodies for chloro-, bromo-, and iododeoxyuridine, as tested in the single labeling and staining procedures, are summarized in Table 1. The Sera-lab antibody showed a high affinity for BrdUrd and CldUrd but no binding to IdUrd. The Becton Dickinson mouse anti-BrdUrd antibody was found t o bind to iodo-, bromo-, and chlorodeoxyuridine but, after washing with Tris buffer, all antibody could be removed form the CldUrd labeled nuclei. No further reduction in the binding of the Becton Dickinson antibody to CldUrd could be reached by incubating the nuclei with the Sera-lab anti-BrdUrd antibody first. Optimal results for double staining were obtained using the rat monoclonal anti-BrdUrd antibody from Sera-lab in a dilution of 1:600 to detect nuclei labeled with CldUrd and the Becton Dickinson antibody in a dilution of 1:2 to detect nuclei labeled with IdUrd. FITC conjugated goat antirat and Texas-Red

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DAPI fluorescence

64

FIG.4. Bivariate IdUrd and CldUrdiDNA distributions from asynchronously growing MOS cells. Cells were first labeled with iododeoxyuridine and 3 h later with chlorodeoxyuridine and subsequently double stained. a. IdUrd was detected with the mouse anti-BrdUrd monoclonal antibody (Becton Dickinson) and Texas-Red conjugated

-

-A-

0

Propidium iodide fluorescence

64

goat antimouse IgG. DAPI was used for the staining of DNA. b. CldUrd was detected with the rat anti-BrdUrd monoclonal antibody (Sera-lab) and fluorescein conjugated goat antirat, IgG. PI was used for the staining of DNA.

DOUBLE STAINING FOR SIMULTANEOUS FCM O F IdUrd AND CldUrd INTO DNA

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procedure when applied to unlabeled MOS cells and MOS cells which were individually labeled with either chloro- or iododeoxyuridine and subsequently double stained. The horizontal axis depicts the IdUrd label (Texas-Red) and the vertical axis the CldUrd label (FITC). No aspecific binding was observed in the unlabeled cells. Figure 3 shows the results of the double labeling, double staining experiments. The MOS cells in this case were pulse labeled with IdUrd, followed 3 h later by a pulse of CldUrd. The double labeled cells that have incorporated both CldUrd and IdUrd are clearly separated from the cells that have incorporated CldUrd (FITC) or IdUrd (Texas-Red) alone. Figure 4 shows that cells that have been double labeled and subsequently have gone through the doubleantibody staining procedure were actively engaged in DNA synthesis. The effect on the cell cycle distribution of the double labeling and double staining procedure is shown in Figure 5. No preferential cell loss was observed. The percentages of G1-, S-, G2M-phase cells in unlabeled and in double labeled, double stained cells were 25%, 42%, 38%, and 23.5%, 41%, 35%, respectively. Cell loss was minimal (average 15%) and the same for the single labeling and staining procedure a s for the double labeling and double staining procedure. Experiments with V79 cells gave similar results.

DISCUSSION In this communication we have presented the results 01 obtained with a n indirect fluorescence double staining procedure for cells labeled with two different halogenated deoxyuridines. Because none of the tested monoclonal antibodies was found to bind to one halogenated deoxyuridine exclusively, we had to develop a staining procedure that effectively exploits other differences in binding characteristics of the available antibodies. The Sera-lab anti-BrdUrd antibody showed no binding to IdUrd. The Becton Dickinson mouse anti-BrdIrd antibody was found to bind to iodo-, bromo-, and chlorodeoxyuridine but, after washing with Tris buffer with a high salt concentration the monoclonal antibody, was 0 64 still present in BrdUrd and IdUrd labeled cells, Propidium iodide fluorescence whereas all antibody was removed from the CldUrd FIG. 5. DNA histograms. a. Unlabeled asynchronously growing labeled cells. Background activity was negligible, as MOS cells. b. Double labeled, double stained asynchronously growing can be seen in Figures 2, 3, and 4. Incubation of the MOS cells. cells with the Sera-lab antibody first and then with the Becton Dickinson antibody, followed by washing with Tris buffer, made no difference in the binding of the conjugated goat antimouse polyclonal antibodies from Becton Dickinson antibody to CldUrd. The application of fluorochrome stained antibodies Jackson were used a s second-step antibodies. As can be seen in Figure 1, no cross-reactivity occurred between raised against mouse and rat IgG, respectively, effecthe mouse anti-BrdUrd monoclonal antibody (Becton tively prevented cross-reactivity in the second step of Dickinson) and the fluorescein conjugated goat antirat the procedure see (Fig. 1).This is also illustrated in IgG or between the rat anti-BrdUrd monoclonal anti- Figure 3, where the double labeled cells t h a t have both body (Sera-lab) and the Texas-Red conjugated goat an- IdUrd and CldUrd (Texas-Red, FITC) and the single labeled cells are located in different regions of the twotimouse IgG antibodies. Figure 2 shows the results of the double staining dimensional fluorescence distribution. Cell loss was c

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minimal. and the Drocedure did not result in a Dreferential cell loss of one of the cell cycle subcompartments. We expect that this technique will find many new applications in studies of cell cycle kinetics. For instance, it will be possible to measure the rate of entry or exit of cell populations from distinct cycle subcompartments, but one could also study in more detail the effect of cell cycle perturbing agents used in particular in chemotherapy of cancer.

ACKNOWLEDGMENTS We thank Mrs. M. Bitterlin of Becton Dickinson for kindly providing the mouse anti-BrdUrd antibody, Dr. A. Floor of Sanbio B.V. and K. Page of Sera-lab (Great Britain) for kindly providing the r a t anti-BrdUrd antibody, Dr. G.W. Barendsen for his suggestions concerning the manuscript, and Mrs. R. Groot for typing the manuscript.

LITERATURE CITED Bakker PJM, Aten JA, Tukker CJ, Barendsen GW, Veenhof CHN: Flow cytometric analysis of experimental parameters for the immunofluorescent labeling of BrdUrd in various tumour cell lines. Histochemistry 91:425-429, 1989. Begg AC, McNally NJ, Schrieve DC, Karcher H: A method to measure the duration of DNA synthesis and the potential doubling time from a single sample. Cytometry 6:620-626, 1985. Begg AC, Moonen L, Hoflan I, Dessing M, Bartelink H: Human tumour cell kinetics using a monoclonal antibody against iododeoxyuridine: Intratumoural sampling variations. Radiother Oncol 11:337-347, 1988. Beisker W, Dolheare F, Gray JW: An improved immunocytochemical procedure for high-sensitivity detection of incorporated bromodeoxyuridine. Cytometry 8235-239, 1987. Dolbeare F, Gratzner H, Pallavicini MG, Gray J W Flow cytometry measurement of total DNA content and incorporated bromodeoxyuridine. Proc Natl Acad Sci USA 805573-5577, 1983. Dolbeare F, Beisker W, Pallavicini MG, Vanderlaan M, Gray JW: Cytochemistry for bromodeoxyuridine, DNA analysis: Stoichiometry and sensitivity. Cytometry 6521-530, 1985. Dolbeare F, Kuo WL, Vanderlaan M, Gray JW: Cell cycle analysis

by flow cytometric analysis of the incoruoration of iododeoxvuridine (IdUrd) and bromodeoxyuridine (BrdUrd). Proc Am k s o c Cancer Res 29:1896, 1988. 8. Gratzner H: Monoclonal antibody against 5-hromo- and 5 iododeoxyuridine: A new reagent for detection of DNA replication. Science 218:474-475, 1982. 9. Hoshino T, Nagashima T, Cho KG, Muovic JA, Hades J E , Wilson CB, Edwards MSB, Pitts LH: S-phase fraction of human brain tumour in situ measured by uptake of bromodeoxyuridine. Int J Cancer 38:369-374, 1986. 10. Karcher H, McNally NJ, Wilson G, Karcher KH, Prafgner R: In-viva Markierung menschliche Tumoren mit BudR zur fluorozytometrische Bestimmung zellkinetische Parameter. Strahlenther Onkol 163:195-200, 1987. 11. Kikuyma S, Kubota T, Watanabe M, Ishibi K, Abe 0: Cell kinetic study of human carcinomas using bromodeoxyuridine. Cell Tissue Kinet 21:15-20, 1988. 12. Kipp JBA, Jongsma APM, Barendsen GW: Cell cycle phase durations derived by a flow cytofluorometric method using the mitotic inhibition vinblastine. In: Laerum OD (ed): Flow Cytometry IV Proceedings of the IVth International Symposium on Flow Cytometry, Voss Norway (1979), Suppl Acta Pathol Microbiol Scand 341-344, 1980. 13. Riccardi A, Danova M, Wilson G, Uci G, Dormer P, Mazzini G, Brugnatelli S, Gironi M, McNally NJ, Ascari E: Cell kinetics in human malignancies studied with in-vivo administration of bromodeoxyuridine and flow cytometry. Cancer Res 48:6238-6245, 1988. 14. Shihui S, Hoshino T, Vanderlaan M, Gray JW: Double labeling with Iodo- and bromodeoxyuridine for cell kinetic studies. J Histochem Cytochem 37:1007-1011, 1989. 15. Schutte B, Reynders MMJ, van Assche CLMVJ, Hupperets PSGJ, Bosman FT. Blijham (GH): An improved method for the immunocytochemical detection of bromodeoxyuridine labeled nuclei using flow cytometry. Cytometry 8:372-376, 1987. 16. Vanderlaan M, Watkins B, Thomas C, Dolbeare F, Stanker I: Improved high affinity monoclonal antibody to iododeoxyuridine. Cytometry 4:499-507, 1986. 17. Wilson GD, McNally NJ, Dunphy E, Karcher H, Pragner R The labeling index of human and mouse tumors assessed by bromodeoxyuridine staining in vitro and in vivo and flow cytometry. Cytometry 6:641-647, 1985. 18. Wilson GD, McNally NJ, Dische S, Saunders MI, Des Rochers C, Lewis AA, Bennett MH: Measurements of cell kinetics in human tumours in vivo using bromodeoxyuridine incorporation and flow cytometry. Br J Cancer 58:423-431, 1988.

An indirect immunofluorescence double staining procedure for the simultaneous flow cytometric measurement of iodo- and chlorodeoxyuridine incorporated into DNA.

In this paper we describe an indirect fluorescence double staining procedure for the simultaneous detection of IdUrd and CldUrd in the same cell nucle...
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