Danish Society for Flow Cytometry
21th
Meeting -
Detection
of Intracellular Antigens by Flow Cytometry
Location: Store
Auditorium,
Organizer:
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,
14:15-15:00
Andreas Thiel,
15:00-15.15 Mogens
H. Claesson & Søren Bregenholt,
15:15-15:35 Inge
Marie Svane, Dep. of Oncology / The Stem
Cell Laboratory,
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.
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.
Abstracts
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,
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,
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,
17. Lan, H.Y.,
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
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
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.