Danish Society for Flow Cytometry

                      21th Meeting - Thursday, May 20, 1999


Detection of Intracellular Antigens by Flow Cytometry

Location:        Store Auditorium, Institute of Pathology, Odense University, J.B. Winsløwsvej, 5000 Odense C.

Organizer:      Bjarne Møller, Dept. of Clinical Immunology, Odense University Hospital. DK-5000 Odense C, Denmark. Phone: (45) 6541 3576. Fax: (45) 6612 7975. E-mail: bjarne.moeller@ouh.dk


12:30-12:35    Wellcome (Bjarne Møller, DSFCM)

12:35-13.20    János Kappelmayer, Department of Clinical Biochemistry and Molecular Pathology, University Medical School, Debrecen, Hungary: Comparative evaluation of different clones and permeabilization techniques for the identification of intracellular differentiation antigens.

13:20-13:35    Jørgen K. Larsen, Finsen Laboratory, Rigshospitalet, Copenhagen University Hospital: Methods for flow cytometric analysis of cell proliferation.

13:35-13:50    Ye Liang & Carsten Röpke, Institute of Medical Anatomy, University of Copenhagen: Flow cytometric detection of some proteins involved in the apoptotic cascade.

13:50-14:15    Coffee break.

14:15-15:00    Andreas Thiel, Deutsches Rheuma-Forschungszentrum, Berlin, Germany: FCM cytokine studies: detection and quantification.

15:00-15.15    Mogens H. Claesson & Søren Bregenholt, Institute of Medical Anatomy, University of Copenhagen: Detection of intracellular cytokines in lymphocytes.

15:15-15:35    Inge Marie Svane, Dep. of Oncology / The Stem Cell Laboratory, Herlev Hospital, University of Copenhagen. Evaluation of immune reactivity in cancer patients during high-dose chemotherapy: Antigen specific activation of low-frequency T-lymphocyte subpopulations as measured by fast immune cytokine assay.

15:35-15:55    Søren Bregenholt, Department of Medical Anatomy, University of Copenhagen, and INSERM U429, Hôpital Necker-Enfants Malades, Paris, France. The use of flow cytometry to dissect intestinal CD4+ T-cell function in experimental inflammatory bowel disease.

15:55-16:15    Claus Munck Petersen, Institute of Medical Biochemistry, University of Aarhus: Processing and Sorting of Sortilin.

16:15-16:30    Coffee break.


16:30-            General assembly of the Danish Society for Flow Cytometry.

                      (Dagsorden, se DSFCM's Nyhedsbrev af 15. april 1999)

                      All are welcome!

                      DSFCM greatly acknowledges the following sponsors supporting this meeting:

                      Becton-Dickinson , Ramcon A/S & DAKO A/S.




Comparative evaluation of different clones and staining techniques for the
identification of intracellular hemopietic antigen

Janos Kappelmayer, Department of Clinical Biochemistry and Molecular Pathology, University
Medical School,
Debrecen, Hungary

Detection of intracellular markers is essential for the proper
identification of acute hematological malignancies. In a multi-center study
we attempted to establish the utility of the commercially available
intracytoplasmic staining techniques for the three basic markers of the
myeloid, B- and T-lineages in normal samples and in acute leukemias (n=21).
Twelve antibodies derived from seven clones labeled with FITC and PE
against myeloperoxidase (MPO), CD3 and CD79a were cross evaluated in a
triple color staining method by using six different intracytoplasmic
techniques. All techniques were suitable for the identification of the
above markers but with largely different efficiency. Permeacyte (Bio-E)
significantly altered scatter properties of cells of normal samples as well
as leukemic blasts and made it impossible to reliably identify leukocyte
subsets on FS-SS or CD45-SS plots. Permeafix (Ortho) resulted in difficulty
in differentiating neutrophils and monocytes on scatter plots.
Cytofix/Cytoperm (Pharmingen) caused a significant increase in
autofluorescence on both FITC and PE channels that resulted in unfavourable
signal to noise ratios compared to other techniques. In case of MPO, PE
conjugates were more effective than FITC conjugates in labeling both normal
myeloid cells or myeloid blasts. Net fluorescence intensities were highest
with MPO-7 clone followed by CBL-MPO-1 and H-43-5. It was found that CD79a
antibodies derived from the same HM47 clone were equally efficient. Out of
the CD3 antibodies the most effective was the UCHT-1 clone while the clone
Hit3a was by far the least sensitive for the identification of early
T-cells. Several fixation/permeabilization protocols (Fix and Perm,
Intraprep, Intrastain, Permeafix) exist that allow reliable and sensitive
detection if intracellular MPO, CD79a and CD3 in normal and leukemic
cells. However special attention should also be paid to the monoclonal
antibody clones and their fluorochrome conjugates since different results
may be obtained regarding sensitivity or even specificity once combined
with a particular intracytoplasmic protocol.

Methods for flow cytometric analysis of cell proliferation

Jørgen K. Larsen, Finsen Laboratory, Finsen Center, Rigshopitalet, Copenhagen University Hospital

1) Cell cycle distribution

Flow cytometric analysis of the nuclear DNA content, using the dye propidium iodide (1) or alternatively Hoechst 33342, DAPI, 7-amino actinomycin D, or To-Pro-3, reveals the distribution of cells in the G0/1, S, and G2+M phases (1). Due to its metachromatic nature, acridine orange enables the discrimination of quiescent G0 cells (2).

The distribution between cycling and non-cycling cells may be estimated by immunochemical staining of the cell proliferation associated antigens PCNA or Ki-67. Double-staining of PCNA and Ki-67 enables dicrimination of the G0, G1, S, G2 and M phases (3). Further mapping of the cell cycle is possible using double-staining of DNA and different cyclins. The distribution of cyclin B1 together with DNA enables distinction between G2+M cells from a lower polyploidization step and G0/1 cells from a higher polyploidization step (4; 5).

Direct etimates of cell cycle progression is not possible with these techniques, even though the perturbations of the cell cycle distribution that may be measured from a time series of measurements may form a basis for indirect estimates of kinetic parameters (6).

2) Cell kinetic measurements

Complete determination of the rate of cell production by division as well as the duration of the cell cycle and its various phases (7) is based on the following techniques: a) Labelling of the DNA synthesizing cells with bromodeoxyuridine (BrdUrd) by a pulse-chase or a continuous labelling procedure, which may be extended to a double-labelling with IdUrd and CldUrd (8-10); b) Mitotic arrest uing stathmokinetic agents such as colcemide and a method for discrimination of the mitotic cells from interphase cells (11; 12). For the classical "percent labelled mitosis" method these two techniques are combined (7; 13).

A quite different approach is to label the cell membrane with a fluorescent molecule and then measure the dilution of this label due to cell divisions (14).

3) Methods for detection of cell proliferation markers

For flow cytometric analysis of intracellular antigens it is necessary to make the cells permeable to specific antibodies, so that these and appropriate dye molecules can reach the respective antigens in the cell interior. At the same time, the antigens must be preserved in their natural, antigenic conformation, and leakage out of the cell must be prevented. It is evident that no single protocol for cell preparation and staining is generally applicable. For every new intracellular antigen or different cell type to be investigated, an appropriate staining method has to be optimized experimentally. However, a series of methods for permeabilization and fixation that works for staining and analysis of a variety of types of antigens are available as a first line of methods to be tested for the particular situation (15-17).

BrdUrd that has been incorporated into DNA can be flow cytometrically detected either by measuring the quenching of fluorescence from the AT-DNA specific dye Hoechst 33342 (Poot), or by immunocytochemical staining with an anti-BrdUrd antibody. The latter is only possible after the DNA has been partially denatured to the single-stranded state using treatment with HCl, HCl/pepsin, DNase-1, or DNA restriction enzymes (8), or by selective DNA strand break induction by photolysis (SBIP) (18). Double-labelling with IdUrd and CldUrd can be matched with antibodies specific for each of these halogenated deoxyuridines (8).

4) Multiparameter studies

With the 488 nm argon laser excitation, DNA content may be measured together with one or two antigens, using staining with FITC-conjugated antibody and propidium iodide, or with FITC- and R-phycoerythrin-conjugated antibodies and 7-amino actinomycin D (5; 19).

Immunochemically, BrdUrd may be measured in a bivariate analysis together with DNA, using denaturation with HCl or HCl/pepsin, (8; 13), or together with another antigen, in this case DNA denaturation with DNase-1 or restriction enzymes may be preferable (20; 21). In a trivariate analysis BrdUrd can be measured immunochemically together with DNA and e.g. cytokeratin (19), together with DNA and a cell surface antigen (22), or together with two cell surface antigens (20; 21).

For the quenching method, based on staining with Hoechst 33342 and ethidium bromide, an ultraviolet light source is necessary. Using ultraviolet light in combination with 488 nm light, this can be extended to a multivariate analysis with measurement of Hoechst 33342 and 7-amino actinomycin D together with FITC- and R-phycoerythrin-conjugated antibodies (23; 24).

1.     Vindelov, L.L. and Christensen, I.J. A review of techniques and results obtained in one laboratory by an integrated system of methods designed for routine clinical flow cytometric DNA analysis. Cytometry, 11: 753-770, 1990.

2.     Darzynkiewicz, Z. Simultaneous analysis of cellular RNA and DNA content. Methods Cell Biol., 41: 401-420, 1994.

3.     Landberg, G. and Roos, G. Flow cytometric analysis of proliferation associated nuclear antigens using washless staining of unfixed cells. Cytometry, 13: 230-240, 1992.

4.     Darzynkiewicz, Z., Gong, J., Juan, G., Ardelt, B., and Traganos, F. Cytometry of cyclin proteins. Cytometry, 25: 1-13, 1996.

5.     Darzynkiewicz, Z., Gong, J., and Traganos, F. Analysis of DNA content and cyclin protein expression in studies of DNA ploidy, growth fraction, lymphocyte stimulation, and the cell cycle. Methods Cell Biol., 41: 421-435, 1994.

6.     Mortensen, B.T., Hartmann, N.R., Christensen, I.J., Larsen, J.K., Kristensen, T., Wieslander, S.B., and Nissen, N.I. Synchronization of the human promyelocytic cell line HL 60 by thymidine. Cell.Tissue.Kinet., 19: 351-364, 1986.

7.     Aherne, W.A., Camplejohn, R.S., and Wright, N.A. An introduction to cell population kinetics. London: Edward Arnold, 1977.

8.     Dolbeare, F. Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part I: Historical perspectives, histochemical methods and cell kinetics. Histochem.J., 27: 339-369, 1995.

9.     Dolbeare, F. Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part II: Oncology, chemotherapy and carcinogenesis. Histochem.J., 27: 923-964, 1995.

10.   Dolbeare, F. Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part III. Proliferation in normal, injured and diseased tissue, growth factors, differentiation, DNA replication sites and in situ hybridization. Histochem.J., 28: 531-575, 1996.

11.   Darzynkiewicz, Z., Traganos, F., and Kimmel, M. Assay of cell cycle kinetics by multivariate flow cytometry using the principle of stathmokinesis. In: J.W. Gray and Z. Darzynkiewicz (eds.), Techniques in cell cycle analysis , pp. 291-336, Clifton, New Jersey, USA: Human Press. 1987.

12.   Larsen, J.K., Munch Petersen, B., Christiansen, J., and Jorgensen, K. Flow cytometric discrimination of mitotic cells: resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde. Cytometry., 7: 54-63, 1986.

13.   Jensen, P.O., Larsen, J.K., Christensen, I.J., and van, E.P. Discrimination of bromodeoxyuridine labelled and unlabelled mitotic cells in flow cytometric bromodeoxyuridine/DNA analysis. Cytometry, 15: 154-161, 1994.

14.   Horan, P.K., Melnicoff, M.J., Jensen, B.D., and Slezak, S.E. Fluorescent cell labeling for in vivo and in vitro cell tracking. Methods Cell Biol., 33:469-90: 469-490, 1990.

15.   Bauer, K.D. and Jacobberger, J.W. Analysis of intracellular proteins. Methods Cell Biol., 41:351-76: 351-376, 1994.

16.   Larsen, J.K. Measurement of cytoplasmic and nuclear antigens. In: M. Ormerod (ed.), Flow cytometry. A practical approach, pp. 93-117, Oxford, UK: Oxford University Press. 1994.

17.   Lan, H.Y., Hutchinson, P., Tesch, G.H., Mu, W., and Atkins, R.C. A novel method of microwave treatment for detection of cytoplasmic and nuclear antigens by flow cytometry. J.Immunol.Methods, 190: 1-10, 1996.

18.   Li, X. and Darzynkiewicz, Z. Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation. Cell Prolif., 28: 571-579, 1995.

19.   Schutte, B., Tinnemans, M.M., Pijpers, G.F., Lenders, M.H., and Ramaekers, F.C. Three parameter flow cytometric analysis for simultaneous detection of cytokeratin, proliferation associated antigens and DNA content. Cytometry, 21: 177-186, 1995.

20.   Penit, C. and Vasseur, F. Phenotype analysis of cycling and postcycling thymocytes: evaluation of detection methods for BrdUrd and surface proteins. Cytometry, 14: 757-763, 1993.

21.   Carayon, P. and Bord, A. Identification of DNA-replicating lymphocyte subsets using a new method to label the bromo-deoxyuridine incorporated into the DNA. J.Immunol.Methods., 147: 225-230, 1992.

22.   Holm, M., Thomsen, M., Hoyer, M., and Hokland, P. Optimization of a flow cytometric method for the simultaneous measurement of cell surface antigen, DNA content, and in vitro BrdUrd incorporation into normal and malignant hematopoietic cells. Cytometry, 32: 28-36, 1998.

23.   Kubbies, M. H . In: Anonymous1999.

24.   Landberg, G. and Roos, G. Proliferating cell nuclear antigen and Ki-67 antigen expression in human haematopoietic cells during growth stimulation and differentiation. Cell Prolif., 26: 427-437, 1993.


Flow cytometric detection of Bc1-2 family proteins involved in the apoptotic casade

Ye Liang & Carsten Röpke, Institute of medical Anatomy, University of Copenhagen

The Bcl-2 family of proteins plays a pivotal role in regulating cell life and death. Many of these proteins reside in the outer mitochondrial membrane, oriented towards the cytosol. Cytoprotective Bcl-2 family proteins such as Bcl-2 and Bcl-XL prevent mitochondrial permeability transition pore opening and release of apoptogenic proteins from mitochondria under many circumstances that would otherwise result in either apoptosis or necrosis . In contrast, some pro-apoptotic members of this family such as Bax can induce these destructive changes in mitochondria. We have investigated intracellular Bcl-2, Bcl-XL and Bax expression in cultured retinal pigment epithelium (RPE) during UV-A induced apoptosis.

Method for detection of Bc1-2 family proteins:

Trypsinize RPE celle, wash with PBS.

Fix with 2% paraformaldehyde for 10 min on ice.

Wash with PBS.

Block the unspecific binding by 10% FCS in 0.2% saponin for lO min at RT.

Label with primary Ab in staining buffer* for 30min on ice.

Wash with 2% FCS in PBS.

Label with fluorescent secondary Ab in staining buffer in the dark for 30 min on ice.

Wash with 2% FCS in PBS.

Analyse by flow cytometry.

* Staining buffer: 2% FCS in 0.2% saponin/PBS.

For each protein we tested, controls were: fixative control, surface binding, primary Ab negative control, secondary Ab negative control.

By the above method we detected the Bcl-2, Bcl-XL and Bax protein expression in RPE cells after W-A exposure and compared this to induced apoptosis. In addition, we compared these protein expressions in RPE cells grown on different supports: ECM coated dishes and uncoated plastic dishes. Some results from these experiments will be shown, and it is concluded that the use of this method for detection of intracellular proteins make it possible to obtain reliable results of expression of the Bcl-2 family proteins - expressions which correlates to apoptotic indices.


The use of flowcytometry to dissect intestinal CD4+ T-cell function in
experimental inflammatory bowel disease.

Soren Bregenholt, Department of Medical Anatomy, University of Copenhagen,
and INSERM U429, Hopital Necker-Enfants Malades, Paris, France.

A chronic and lethal inflammatory bowel disease (IBD), can be induced in
immunodeficient (SCID) mice, by adoptive transfer of CD4+ T-cells from
syngenic, immunocompetemt donors (1). We have used various flowcytometric
techniques to characterize the lamina propria infiltrating CD4+ T-cells
from SCID with IBD.
When staining for a panel of intracellular cytokines, we found a large
increase in the numbers of CD4+ T-cells producing IFN-g and TNF-a. A
significant increase in the number of IL-2 producing T-cells could was only
found in mice with severe pathological changes. Conversely, IL-10 producing
CD4+ T-cells were virtually absent from SCID mice with IBD whereas no
changes in the numbers of IL-4 producing cells were observed (2). A similar
increase in the fraction of Th1-like CD4+ T-cells was found in the spleen of
diseased mice (3).
To assess the in vivo proliferation of intestinal CD4+ T-cells these were
labeled by bromo-deoxy-uridine (BrdU) and enumerated by flowcytometry ex
vivo. These experiments show that 5 time more CD4+ T-cells enter the cell cycle
in these mice than in control mice. DNA staining revealed that this was
balance by an increase in the number of apoptotic CD4+ T-cells. By way of CD4,
BrdU, and Annexin-V triple staining is was shown that the apoptotic cells were
all derived from the pool of expanding CD4+ T-cells (4).
To specifically study the role of IFN-g in IBD, the disease was induced in
SCID mice by transplanting CD4+ T-cells from IFN-g deficient donors. These
experiment revealed that the IFN-g deficient cells had retained their
capacity to produce TNF-a and IL-2. Surprisingly, a 2-3 fold increase in the
fraction of IL-4 producing CD+ T-cells was found in these mice when compared to SCID
mice transplanted with WT cells. Analysis of in vivo proliferation by
BrdU-incorporation showed that the number of proliferating cells in these
mice were increased by two fold when compared to normal mice, although they were
reduced as compared to WT transplanted mice. This was probably due to the
impaired ability of the transplanted T-cells to up-regulate MHC-II expression
on epithelial cells as assess by ex vivo flowcytometry. A central role for
IL-12 in driving the IFN-g production by CD4+ T-cells in IBD, was shown by the
impaired ability of IL-12-unresponsive STAT-4 deficient CD4+ T-cells to
produce IFN-g, but retaining their ability to produce both TNF-a and IL-2 (5).
These data point towards an essential role for a IL-12-IFN-g-MHC-II-axis in
the induction of CD4+ T-cell activation and eventually IBD.

1 Claesson MH, et al. Clin.Exp.Immunol. 1996; 104:491-500.

2 Bregenholt S and Claesson MH. Eur.J.Immunol. 1998; 28:379-389.

3 Bregenholt S and Claesson MH. Clin.Exp.Immunol. 1998; 111:166-173.

4 Bregenholt S, Reimann J, Claesson MH.. Eur.J.Immunol. 1998; 28:3655-3663.

5 Claesson MH, Bregenholt S, Bonhagen K, et al. J.Immunol. 1999; 162:3702-3710.


Processing and Sorting of Sortilin

Claus Munck Petersen, Dept. of Medical Biochemistry, University of Aarhus.

We have previously reported [1] the purification and sequencing of
sortilin, a type I membrane-receptor, with similarities to known sorting
receptors, e.g.the mannose-6-phosphate receptors and yeast Vps10p. Sortilin
is expressed in several tissues and is particularly abundant in brain,
testes and skeletal muscle.
Recent findings [2] show that sortilin is synthezised as a
non-ligand-binding precursor molecule which is activated by propeptide
cleavage in distal parts of the synthetic pathway. Activated sortilin binds
Lipoprotein Lipase (LpL) [3], neurotensin [4,2] and the ER-resident
receptor associated protein (RAP) [1,2]. Sortilin's cytoplasmic domain
contains several putative sorting segments, and results obtained by means
of hybrid receptors (containing the sortilin tail) stably expressed in
different cell types suggest that the receptor conveys intracellular
sorting, including Golgi-endosome transport, of its ligands.

1. Petersen, CM et al. (1997), J. Biol. Chem., 272: 3599;

2.Petersen, CM et al. (1999), EMBO J., 18: 595;

3. Nielsen MS et al. (1999), J. Biol. Chem., 274: 8832;

4. Mazella, J et al. (1998), J. Biol. Chem., 273: 26273.