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Cell Function in Cytomics

functional flow cytometry, functional cytometry, functional cytomics

former: Cell Biochemistry Group
*external link Max-Planck-Institut für Biochemie, Martinsried

G.Valet





Table of Contents




1. CELLS AND DISEASE

The measurement of cellular parameters (cytometry) is of significant importance for medical purposes because diseases are caused by biochemical changes in cellular systems or organs.

Although this fact is generally acknowledged, the majority of the biochemical analysis in human disease is presently not performed on cells but rather on blood plasma or serum, urine, liquor cerebrospinalis, tissue extracts etc.


2. Cell Structure and Cell Function
2.1 Structural Cytometry (Structural Cytomics):

The cytometric determination of structural cell constituents (tab.1, 1/ge) is in use for many years e.g. for the determination of DNA distributions, cellular antigen contents (7/da, 5/da, 4/da, 2/da, 1/da, 14/cl, 12/cl, 11/cl, 10/cl, 9/cl, 8/cl, 7/cl) or natural pigment analysis like chlorophyll or phycobiliproteins in plankton cells (1/pl). Simultaneous biophysical measurements of electrical or optical (FSC) cell volume and optical granularity (SSC) complement the cell characterization.


2.2 Functional Cytometry (Functional Cytomics):

Structural flow cytometric measurements do usually not permit to follow rapid functional changes of cells such as e.g. changes of intracellular pH, of cellular excitation or of energy production upon stimulation.

Functional changes are early indicators of cell growth, death or differentiation e.g. in clonal development of the immune or hematopoietic system but they equally indicate signalling for specific actions of differentiated cells e.g. granulocytes under cytokine influence or thrombocytes in prethrombotic conditions.

Comparing a cell for a moment with a house: when somebody puts on his coat and takes an umbrella, a functional but no structural change has occured since neither the number of persons, coats or umbrellas have changed. The recognition (diagnosis) of this functional change permits the prediction that somebody will soon leave the house.

The extraction of predictive information from cell function measurements is e.g. of high interest for predictive medicine but also for cell biological or environmental research in general.


2.3 Cytomics and Medical Bioinformatics (Data pattern Classification)

Flow and image cytometric measurements provide multiparametric data. Due to the high amount of data, the usual analysis procedures extract mostly only a few elementary parameters e.g. % frequency or concentration of a certain cell type in the peripheral blood i.e. the majority of result information is not evaluated due to a lack of easily applicable methodology.

While typical multiparameter information extraction methods like neural network, cluster or principal component analysis, expert systems, fuzzy logic or statistical classifiers have for a variety of reasons mostly not reached a practicable stage for widespread medical applications, a new data pattern classification algorithm seems more promising in this respect. Standardized i.e. interlaboratory portable multiparameter data classifiers are obtained, no mathematical assumptions are required, the selected classification parameters are intuitive and the classifiers can be learned as well as applied on typical personal computers (PC).


2.4 Applications in Research and Medicine:

The combination of cytomics with medical bioinformatics (15/cl, 14/cl, 13/cl, 12/cl, 11/cl, 10/cl, 9/cl, 8/cl, 7/cl, 5/cl, 4/cl, 2/cl, 7/da, 6/da, 5/da, 4/da, 3/da, 2/da, 1/da) provides an efficient new strategy for the standardized expression of molecular disease diagnosis, for predictions of disease development in the individual patient as well as for the control of therapeutic efficiency in clinical medicine (Clinical Cytomics, Medical Cytomics). Cell biochemical, clinical chemistry or other clinical parameters can be classified by this standardized multiparameter data classification (SMDC). Efforts for the practical implementation of this approach are under way e.g. in the European Working Group for Clinical Cell Analysis (EWGCCA).


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3. Cell function:
3.1 Microscopical methods:

The determination of single cell functions was difficult and limited in the past because microelectrodes had to be introduced into single cells e.g. for transmembrane potential or pH-measurements. More recently patch clamping techniques have enlarged the potential for functional investigations on cells or membrane fragments but only the development of laser scanning microscopes, digital imaging techniques and appropriate fluorescent sensor (indicator) dyes has fully opened the large potential of cell function measurements in recent years.

The advantage of the microscope consists in the detailed morphological single cell observation. Disadvantages concern difficulties in quantitation, usually lower speed of analysis, prolonged sample exposure to high light intensities resulting in fluorescence bleaching, sample heating and observation of cells in the very small fluid volume under the cover slip under potentially suboptimal conditions of osmotic pressure, nutrient or oxygen/CO2 concentrations. Prolonged exposure to high light intensities may in addition influence the biochemical behaviour of cells.


3.2 Flow cytometric methods:

Cell functions ( tab.1) can be determined at high speed in flow cytometers with a multitude of biochemically specific, non toxic fluorescent indicator molecules close to the in-vivo state and without prolonged exposure to unusual light levels. Heterogeneous cell suspensions from the body but also from sweet and salt water microorganism suspensions can be investigated with a minimum of preparation.

The acceleration in the flow cytometer and the transition of a cell through the sensing light beam takes only fractions of a second (5-50usec) i.e. the influence of the measurement process on the cells is minimal. Furthermore flow cytometry offers ideal statistical sampling as well as optimal biochemical conditions during the pre-measurement as well as during the short measurement phase. This mostly outweighs the usual lack of high spatial resolution as well as the impossibility to investigate the same cell several times e.g. with time or following restaining with other biochemical markers which represent typical advantage of the microscope.

Functional cell assays are easily performed by flow cytometry. The addition of dye or dye cocktail is followed by an incubation time between 1 and 15 min at room temperature or 37C. In most instances the stained cell sample can then be measured immediately i.e. without centrifugation. In some instances a dilution or a centrifugation step followed by resuspension of the cells in buffer is required to lower the extracellular dye concentration (e.g. rhodamine123). The centrifugation step can, however, mostly be substituted by a dilution step if the primary assay is performed in a small buffer volume at high cell concentration.


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tab.1
CELL BIOCHEMISTRY (CELLULAR BIOCHEMISTRY) BY FLOW CYTOMETRY

STRUCTURE
- DNA/RNA
- proteins
- carbohydrates
- lipids
- antigens
- hormon receptors
- fluorescent in-situ hybridization
FUNCTION
- metabolism: intracellular pH
- excitation: calcium
- energy: transmembrane and mitochondrial potentials
- oxidation: O2-,H2O2, free radicals
- reduction: glutathione, free protein SH-groups
- enzymes: esterases, peroxidases, proteases (cathepsins), glucosidases, phosphatases etc.

4. Flow cytometer setup for functional stains: 4.1 mammalian cells:

Using the 488nm line of an argon ion laser for fluorescence excitation, the emitted fluorescent light is typically collected by bandpass filters between 510-540nm, 550-590nm and 620-750nm corresponding to the fluorescein (FITC), phycoerythrin (PE) and tandem conjugate (PE-CY5, PERCP) channel for immune pheno typing.

Functional stains are usually equally well measurable with a high pressure mercury arc lamp (HBO-100) using a 450-500nm band pass filter for fluorescence excitation in combination with the above emission filters.

The fluorescence emission light channels can be used for a variety of functional stains e.g. the green FITC-light channel for: DiOC6 (3/fu) transmembrane potential, rhodamine123 (R123) (8/cy, 7/cy, 6/cy, 5/cy) mitochondrial potential, dihydrorhodamine123 (DHR) (14/fu, 11/fu, 10/fu, 8/fu, 6/da, 4/da, 6/cl) or dichlorofluorescin (DCFH) (15/fu, 9/fu, 6/fu, 1/cl) metabolic burst measurements, rhodamine110 (R110) (24/fu, 23/fu, 22/fu, 21/fu, 20/fu, 19/fu, 18/fu, 17/fu, 13/fu, 6/da) substrates for the determination of cysteine-, serine-, metallo- or carboxy proteinases, the orange PE-light channel for FLUO-3 calcium or SNARF-1 intra cellular pH-measurmenents while the red light channel is frequently used to check the presence and DNA-distribution of dead cells following staining with propidium iodide (PI).

The strong UV-lines of high pressure mercury arc lamps around 365nm can be used to excite 1,4-diacetoxy-2,3-dicyanobenzene (ADB) (7/fu, 2/fu, 1/fu, 8/cy, 4/cy, 3/cy, 2/cy, 1/cy, 5/cl, 4/cl, 3/cl, 2/cl, 1/cl) for intracellular pH-measurements, INDO1 (4/fu, 1/cl) calcium stain or orthophtaldialdehyde (OPT) (5/fu) free glutathione and free protein SH-group stain. Fluorescence emission is collected by a 390-440nm bandpass filter and a 500nm long pass filter. The DNA of dead cells is again stained with PI which equally well excites at 365nm and at 488nm.


4.2 Microorganisms and Plant Cells:

Microorganisms and plant cells can be measured in principle like mammalian cells. Microorganism require special care for a sensitive Forward and sideward scatter light detection. Strong autofluorescence from a variety of sources e.g. natural pigments (chlorophyll, phycobiliproteins) may occur i.e. care has to be taken that the specific cellular fluorescence staining is not significantly influenced by autofluorescence.


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5. Data evaluation:

Usually several stains are performed on aliquots of the same cell sample. The measurements are recorded as list mode files (FSC, SSC, F1, F2, F3, time) and can be automatically evaluated by the CLASSIF1 program system. The results of this multi window evaluation in several dimensions are stored in databases.

Given a learning set of clinically or experimentally known cell samples, the software learns the most significant differences from the different databases, merges the data columns of interest into a new database and automatically establishes a self learning classifier.

This classifier can prospectively classify unknown cell samples, stained according to the same rules as the training set samples. Provided that the measurements are properly long term standardized with fluorescent calibration particles, standardized classifiers can be elaborated. They are flow cytometer and laboratory independent and therefore suitable for consensus formation and international standardization.


6. Cytometry on the INTERNET:

Information on new developments can be obtained on Internet from the Martinsried Cell Biochemistry homepage: http://www.classimed.de/cellbio.html or more generally from the
CytoRelay node. The address (URL) is: http://www.classimed.de/cytorel.html

Off-line Internet copies of the entire information on both nodes for use in PC's are obtained by regularly updated downloads from http://www.classimed.de/classim1.zip and http://www.classimed.de/classim2.zip

Historical aspects of flow cytometry concern the emergence of present concepts, as well as developments in specific laboratories or of the entire cytometry discipline.


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7. FUNCTIONAL CELL STAINS FOR FLOW-CYTOMETRY

The majority of the subsequent methods were developed in our laboratory (see 9.Selected Literature References)

7.1 DYES:

ADB (7/fu, 2/fu, 1/fu, 8/cy, 4/cy, 3/cy, 2/cy, 1/cy, 5/cl, 4/cl, 3/cl, 2/cl, 1/cl) = 1,4-diacetoxy-2,3-dicyano-benzene (MW: 244.2), Nr.D-0679, Sigma Chemicals, St.Louis, USA
INDO1/AM (4/fu, 1/cl) = INDO1 pentaacetoxymethyl-ester (MW: 1009.9), Nr.I-3261, Sigma Chemicals, St.Louis, USA Nr.I1203, Molecular Probes, Eugene, USA
OPT (5/fu) = ortho-phthaldialdehyde (MW: 134.1), Nr.P-1378, Sigma, St.Louis, USA
HOECHST 33258 = Bisbenzimide H33258 (MW: 623.97), Nr.B-2883, Sigma Chemicals, St.Louis, USA
HOECHST 33342 = Bisbenzimide H33342 (MW: 615.99), Nr.B-2261, Sigma Chemicals, St.Louis, USA
DIOC6 (3/fu) = 3,3-dihexyl-oxacarbocyanin (MW: 572.53), Nr.14414, Eastman Kodak, Rochester, USA
R123 (8/cy 7/cy, 6/cy, 5/cy) = rhodamine 123 (MW: 380.8), Nr.R-8004, Sigma Chemicals, St.Louis, USA
DCFH-DA (15/fu, 9/fu, 6/fu, 1/cl) = 2',7'-dichlorofluorescin-diacetate, (MW: 487.3), Nr.D-6883, Sigma Chemicals, St.Louis, USA Nr.D-399, Molecular Probes, Eugene, USA
DCF (15/fu, 9/fu, 6/fu, 1/cl) = 2',7'-dichlorofluorescein (MW: 401.2), Nr.D-6665, Sigma Chemicals, St.Louis, USA
DHR123 (14/fu, 11/fu, 10/fu, 8/fu, 6/da, 4/da, 6/cl) = dihydrorhodamine 123 (MW: 346), Nr.D-632, Molecular Probes, Eugene, USA
HE (16/fu, 15/fu, 9/fu, 1/cl) = hydroethidine (MW: 315), Nr.17084, Polysciences, Warrington, USA
SNARF1 = carboxy SNARF1 AM acetate, (MW: 568), Nr.C-1271, Molecular Probes, Inc., Eugene, USA
FLUO3/AM = FLUO-3 AM cell permeant, (MW: 1130), Nr.F-1241, Molecular Probes, Eugene, USA
R110 = rhodamine 110 (MW: 366.8), Nr.136 1468, Eastman Kodak, Rochester, USA, or: Exciton Chemical Co, Dayton, USA
(Z-Ala2)2-R110 (23/fu, 20/fu, 17/fu, 13/fu, 12/fu, 6/da) = cathepsin G serine protease substrate (MW: 882.94), Nr.R-6504, Molecular Probes, Eugene, USA
(Z-Arg2)2-R110 (23/fu, 20/fu, 19/fu, 18/fu, 13/fu, 12/fu, 6/da) = cathepsin B,L cysteine proteinase substrate (MW: 1369.22), NR.R-6501, Molecular Probes, Eugene, USA,
(Z-PheArg)2-R110 (23/fu, 20/fu, 19/fu, 17/fu, 13/fu, 12/fu) = cathepsin B,(L) cysteine proteinase substrate (MW: 1351.18), Nr.R-6502, Molecular Probes, Eugene, USA
(NH2-Leu)2-R110 (24/fu, 22/fu, 21/fu) = aminopeptidase metalloproteinase substrate (MW: 556.7), Nr.R-6509, Molecular Probes, Eugene, USA
(NH2-Phe)2-R110 (24/fu, 22/fu, 21/fu) = aminopeptidase metalloproteinase substrate (MW:624.62), own laboratory synthesis
AO (3/fu, 2/cl, 1/cl) = acridine orange (MW: 301.83), Nr.4539, Polysciences, Warrington, USA
PI = propidium iodide (MW: 668.4), Nr.P-4170, Sigma, St.Louis, USA
fluorescein = (MW: 376.3), Nr.F-6377, Sigma Chemicals, St.Louis, USA


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7.2 REAGENTS:

PMA = phorbol-myristate-acetate (MW: 662.0), Nr.P-8139, Sigma, St.Louis, USA
H2O2 = perhydrol 30% (MW: 34.01), Nr.7210, Merck, Darmstadt, Germany
FMLP = N-Formyl-Met-Leu-Phe chemotactic bacterial peptide (MW: 437.6), Nr.F-3506, Sigma, St.Louis, USA
TNF-alpha = tumor necrosis factor-alpha, recombinant, expressed in yeast, 2x10**7 U/mg (MW: 17000), Nr.T-0157, Sigma, St.Louis, USA
ionomycin = calcium ionophore (MW: 709.0), Nr.407950, Calbiochem, Bad Soden, Germany
A23187BR = calcium ionophore (MW: 602), Nr.100107, Calbiochem, Bad-Soden, Germany
gramicidin D = H+-ionophore (MW: ...), Nr.G-5002, Sigma, St.Louis, USA,
Z-Phe-Ala-CHN2 = cathepsin B,H,L, cysteine proteinase inhibitor (diazo-methyl-ketone, MW 394.4), Nr.1040, Bachem, Heidelberg, Germany
Z-Leu-CHN2 = aminopeptidase, metalloproteinase inhibitor (chloro- methyl-ketone, MW 200.11), Nr.1260, Bachem, Heidelberg, Germany
DFP = di-isopropylfluorophosphate, cathepsin G serine proteinase inhibitor, (MW 181,15), Nr.D08979, Sigma-Aldrich-Chemie, St. Louis, USA
EDTA = 0.2 M pH 7.2 (di-Na EDTA, (MW: 372.25), Nr.8418, Merck, Darmstadt, Germany
CaCl2 = 0.3 M CaCl2.2H2O solution aqua dest. (MW: 110.99), Nr.2382, Merck, Darmstadt, Germany
HgCl2 = mercury-II-chloride (MW: 200.59), Nr.4404, Merck, Darmstadt, Germany
particles = PolybeadTM Carboxylate Microspheres(2.5% Latex-Solids), UV excitable (365nm), Nr.18340 4.5um BB, 488nm FITC-excitable Nr.16592 4.5um, YG Polysciences Ltd., Warrington, USA,
NaCl = Natriumchlorid (MW: 58.44), Nr.6404, Merck, Darmstadt, Germany
HEPES = N-(2-Hydroxyethyl)-piperazine-N'-2-ethane-sulfonic acid (MW: 238.3), Nr.H-3375, Sigmas Chemicals, St.Louis, USA
HBS-buffer = 0.15M NaCl2, 5mM HEPES pH 7.35
PBS-buffer = phosphate buffered saline (0.137M NaCl, 2.7mM KCl, 10mM PO4 pH 7.4 at 25oC), PBS tablets, Nr.P-4417 Sigma Chemicals, St.Louis, USA
BSA = bovine serum albumin (MW: 65000) 1x crystallized, Nr. A-4378 Sigma Chemicals, St.Louis, USA
DMF = di-methylformamide, (MW: 73.10), Nr.D-27054-7, Sigma, St.Louis, USA
DMSO = di-methylsulfoxide, (MW: 78.13), Nr.D-8779, Sigma Chemicals, St.Louis, USA


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7.3 COMPANY ADDRESSES:

Bachem
Lessingstr. 26
D-69115 Heidelberg
Germany
Tel: +49/6221/163091
Fax: +49/61221/21442
E-mail: --

Calbiochem
Lisztweg 1
D-65796 Bad Soden
Germany
Tel: +49/6196/63955
Fax: +49/6196/62361
E-mail: --

Eastman Kodak
LRPD-1001 Lee Road
P.O.Box 92822
Rochester NY 14692-7073
USA
Tel: +1/716/588-4817
Fax: +1/716/722-3179
E-mail: --

Merck
Frankfurter Str. 250
D-64291 Darmstadt
Germany
Tel: +49/6151-72-0
Fax: +49/6151-72-2000
E-mail: --

Molecular Probes
4849 Pitchford Av.
Eugene, OR 97402-9144
USA
Tel: +1/541/465-8300
Fax: +1/541/344-6504
Molecular Probes, Europe
PoortGebouw Rijnsburgerweg 10
NL-2333AA Leiden
The Netherlands
Tel: +31/71-5233-378
Fax: +31/71-5233-419
E-mail: Customer Service and Technical Assistance: 76225.2203@compuserve.com
Internet: Molecular Probes, USA

Polysciences Inc.
400 Valley Road
Warrington, PA 18976
USA
Tel: +1/215/-343-6484
Fax: +1/215/-343-0214
Polysciences Inc. Europe Handelsstr. 3
D-69208 Eppelheim
Germany
Tel: +49/6221-76576
Fax: +49/6221-764620
E-mail: --

Sigma Chemical Company
P.O.Box 14508
St. Louis, MO 63178-9916
USA
Tel: +1/314-771-5750
Fax: +1/314-771-5757
Sigma-Aldrich Chemie GmbH
Grünwalder Weg 30
D-82041 Deisenhofen
Germany
Tel: +49/89/6513-0
Fax: +49/89/6513-1161
E-mail: --
Internet: Sigma-Aldrich, USA Sigma-Aldrich, Germany


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7.4 UV-EXCITATION (365nm) ASSAYS

HBO-100 lamp, HeCd or Ar-Laser


7.4.1 INTRACELLUAR pH/ESTERASE ACTIVITY:

ADB: (7/fu, 2/fu, 1/fu, 8/cy, 4/cy, 3/cy, 2/cy, 1/cy, 5/cl, 4/cl, 3/cl, 2/cl, 1/cl) 1,4-diacetoxy-2,3-dicyanobenzene MW:244.2 D
standard assay:
250ul cell suspension (1x106-107 cells/ml)
+5ul ADB 1mg/ml, PI 2mg/ml cocktail in DMF
- incubate 5min at 22oC
- measure (ADB 20ug/ml (82uM), PI 40ug/ml (60uM))

pH calibration assay:
250ul cell suspension (5x107 cells/ml)
+5ul ADB 1mg/ml, PI 2mg/ml cocktail in DMF
+5ul gramicidine D 1mg/ml in DMSO
+5ul 5% NaN3-solution in HBS buffer
- incubate 10min at 22oC
- dilute 5ul assay aliquots in 250ul portions of 10mM phosphate buffered saline pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 containing 5ul ADB 1mg/ml, PI 2mg/ml cocktail in DMF
- incubate 10min at 22oC
- measure (ADB 20ug/ml (82uM), PI 40ug/ml (60uM), NaN3 0.1%, gramicidin D 20ug/ml)


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7.4.2 INTRACELLULAR CALCIUM:

INDO1/AM: (4/fu, 1/cl) INDO1-acetoxymethylester MW:1009.9 D
- freeze A23187Br or ionomycin ionophore at -85o C or -180o C to prevent decay

assay1: intracellular calcium
250ul cell suspension
+5ul INDO1/AM 1mg/ml (HBO-100), 0.1mg/ml (HeCd laser), PI 2mg/ml cocktail in DMF
- incubate 15min at 22oC
- measure (INDO1 20.2ug/ml (20.2uM), PI 40ug/ml (60uM))

assay2: ionophore + EDTA = extract intracellular calcium
250ul cell suspension
+5ul INDO1/AM 1mg/ml (HBO-100), 0.1mg/ml (HeCd laser), PI 2mg/ml cocktail in DMF
- incubate 15min at 22oC
+5ul A23187Br or ionomycin ionophore 1mg/ml DMF
- incubate 10min at 22oC
+5ul 0.2M EDTA pH 7.35
- incubate 10min at 22oC
- measure (INDO1 20.2ug/ml (20.2uM), PI 40ug/ml (60uM), EDTA 4mM,A23187Br 20ug/ml (33uM)

assay3: ionophore + calcium = set intracellular calcium to 1mM
250ul cell suspension
+5ul INDO1/AM (ester) 1mg/ml(HBO-100), 0.1mg/ml (HeCd laser), PI 2mg/ml cocktail in DMF
- incubate 15min at 22oC
+5ul A23187Br or ionomycin ionophore 1mg/ml DMF
- incubate 10min at 22oC
+5ul 0.3M CaCl2
- incubate 10min at 22oC
- measure (INDO1 20.2.ug/ml (20.2M), PI 40ug/ml (60uM), CaCl2 6mM, A23187Br 20ug/ml (33uM))


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7.4.3 FREE GLUTATHIONE/FREE PROTEIN SH-GROUPS:

OPT: (5/fu) ortho-phthaldialdehyde MW: 134.1 D

assay1: free glutathione/free protein-SH
250ul cell suspension
+5ul OPT 6.70mg/ml in DMF
+5ul PI 2mg/ml in HBS
- incubate 10min at 0oC
- measure (OPT 134ug/ml, (1mM), PI 40ug/ml (60uM))

assay2: Hg2+ blocking of free SH-groups
250ul cell suspension
+5ul HgCl2 27.15mg/ml in HBS
- incubate 5min at 0oC
+5ul OPT 6.70mg/ml DMF
+5ul PI 2mg/ml in HBS
- incubate 10min at 0oC
- measure (OPT 134ug/ml, (1mM), HgCl2 0.54mg/ml (2.7mM), PI 40ug/ml (60uM))


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7.4.4 VITAL DNA STAIN:

Hoechst H33258 bisbenzimide MW: 623.97 D (slow penetration)

assay:
250ul cell suspension
+5ul H33258 0.312mg/ml in HBS
- incubate 30min at 37oC
+5ul PI 2mg/ml in HBS
- incubate 5min at 22oC
- measure (H33258 6.24ug/ml, (10uM), PI 40ug/ml (60uM))


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7.4.5 VITAL DNA STAIN:

Hoechst H33342 Bisbenzimide MW: 615.99 D (fast penetration)

assay:
250ul cell suspension
+5ul H33342 0.308mg/ml in HBS
- incubate 30min at 37oC
+5ul PI 2mg/ml in HBS
- incubate 5min at 22oC
- measure (H33342 6.16ug/ml, (10uM), PI 40ug/ml (60uM))


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7.5 BLUE LIGHT FLUORESCENCE EXCITATION

Argon Laser (488nm), HBO-100 lamp (450-495nm)



7.5.1 CELLULAR ENERGY:

DI0C6: (3/fu) 3,3-dihexyl-oxacarbocyanine(3) MW: 572.5 D, propidium iodide (PI) MW: 376.3 D
transmembrane potential

assay:
250ul cell suspension
+5ul DIOC6 10ug/ml, PI 2mg/ml in dimethylformamide (DMF)
- incubate for 5 min at 22oC
- measure (DiOC6 0.2ug/ml (0.35uM), PI 40ug/ml (60uM))


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7.5.2 CELLULAR ENERGY:

R123: 8/cy, 7/cy, 6/cy, 5/cy) rhodamine123 MW: 380.8 D, mitochondrial membrane potential

assay:
250ul cell suspension
+5ul R123 0.5mg/ml in DMF
- incubate 30min at 37oC
- dilute with 750ul HBS
+20ul PI 2 mg/ml in 10mM HEPES buffered saline pH 7.35 (HBS)
- incubate 5min at 22oC
- measure (R123 10ug/ml (26uM), PI 40ug/ml (60uM))


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7.5.3 METABOLIC STATE:

SNARF1/AM: intracellular pH, SNARF1-acetoxymethylester MW: 568 D
- predilute: 0.568mg/ml (1mM) SNARF1/AM stock solution in DMF daily 1/100 with 2mg/ml PI in HBS (SNARF1/AM 1/100 5.68ug/ml, 10uM)

assay:
250ul cell suspension
+5ul SNARF1/AM 5.68ug/ml, PI 2mg/ml in HBS
- incubate 30min at 37oC
- measure (SNARF1/AM 0.11ug/ml (.2uM), PI 40ug/ml (60uM))


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7.5.4 EXCITATION STATUS:

FLUO3/AM: intracellular Ca2+, FLUO3-acetoxymethylester MW: 1130 D
- predilute: 1.13mg/ml (1mM) FLUO3/AM stock solution in DMF daily 1/50 with 2mg/ml PI in HBS (FLUO3/AM 1/50 22.6ug/ml, 20uM)

assay:
250ul cell suspension
+5ul FLUO3/AM 22.6ug/ml, PI 2mg/ml in HBS
- incubate 30min at 37oC
- measure (FLUO3/AM 0.45ug/ml (0.4uM), PI 40ug/ml (60uM))


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7.5.5 OXIDATIVE BURST AND PEROXIDASE ACTIVITY:

DHR123: (14/fu, 11/fu, 10/fu, 8/fu, 6/da, 4/da, 6/cl) dihydrorhodamine123 MW: 346.0 D
sensitive H2O2 and peroxidase indicator system (8 x DCFH-DA, (15/fu, 9/fu, 6/fu, 1/cl)

- DHR123 stock solution: 1.5mg/ml (4.0mM) in DMF, freeze at: -180oC
- predilute: 5ul DHR123 1.5mg/ml in DMF + 500ul 2mg/ml PI in HBS (DHR123 1/100, 15ug/ml,40uM, PI 2mg/ml 3mM, DMF 1%), freeze at: -180oC
- PMA stock solution: 0.662mg/ml (1.0mM) in DMF, freeze in 5ul portions in 1.5ml Eppendorf conical tubes with snap caps, predilute freshly with 1ml HBS (PMA 5uM, DMF 0.5%)
- FMLP stock solution 1mM (0.437mg/ml) in DMF, freeze in 5ul portions in 1.5ml Eppendorf conical tubes with snap caps, predilute freshely with 1ml HBS (FMLP 5uM, DMF 0.5%)
- TNF-alpha stock solution 30nM (0.5ug/ml) in phosphate buffered saline (PBS) with 10mg/ml BSA obtained by 1/20 dilution of original solution, e.g. 50ul with 950ul PBS/BSA, freeze in 100ul portions in 1.5ml Eppendorf tubes with snap cap
- H2O2: 1/200 (50mM) prediluted from Perhydrol (Merck) with HBS

assay1: spontaneous oxidation (ex-vivo)
250ul cells
+5ul DHR123 15ug/ml, PI 2mg/ml, DMF 1%
- incubate 15min at 22oC
- measure (DHR123 0.3ug/ml (0.86uM), PI 40ug/ml (60uM), DMF 0.02%)

assay2: FMLP stimulation (differential stimulator assay I)
250ul cells
+5ul DHR123 15ug/ml, PI 2mg/ml, DMF 1%
- incubate 15min at 22oC or 37oC
+5ul FMLP 5uM, DMF 0.5% in HBS
- incubate 15min at 22oC or 37oC
- measure (DHR123 0.3ug/ml (0.86uM), FMLP 100nM, PI 40ug/ml (60uM), DMF 0.03%)

assay3: TNF-alpha stimulation (differential stimulator assay II)
250ul cells
+5ul DHR123 15ug/ml, PI 2mg/ml, DMF 1%
- incubate 15min at 22oC or 37oC
+5ul TNF-alpha 30nM in PBS/BSA
- incubate 15min at 22oC or 37oC
- measure (DHR123 0.3ug/ml (0.86uM), TNF-alpha 6nM, PI 40ug/ml (60uM), DMF 0.03%)

assay4: FMLP+TNF-alpha stimulation (differential stimulator assay III)
250ul cells
+5ul DHR123 15ug/ml, PI 2mg/ml, DMF 1%
- incubate 15min at 22oC or 37oC
+5ul FMLP 5uM, DMF 0.5% in HBS
- incubate 5min at 22oC or 37oC
+5ul TNF-alpha 30nM, DMF 0.5% in HBS
- incubate 10min at 22oC or 37oC
- measure (DHR123 0.3ug/ml (0.86uM), FMLP 100nM, TNF-alpha 6nM, PI 40ug/ml (60uM), DMF 0.03%)

assay5: PMA stimulation (positive control)
250ul cells
+5ul DHR123 15ug/ml, PI 2mg/ml, DMF 1%
- incubate 15min at 22oC or 37oC
+5ul PMA 5uM, DMF 0.5% in HBS
- incubate 15min at 22oC or 37oC
- measure (DHR123 0.3ug/ml (0.86uM), PMA 100nM, PI 40ug/ml (60uM), DMF 0.03%)

assay6: peroxidase activity (optional)
250ul cells
+5ul DHR123 15ug/ml, PI 2mg/ml, DMF 1%
- incubate 15min at 22oC or 37oC
+5ul H2O2 (50mM) prediluted 1:200 with HBS from Perhydrol (Merck)
- incubate 10min at 22oC or 37oC
- measure (DHR123 0.3ug/ml (0.86uM), H2O2 (1mM), PI 40ug/ml (60uM), DMF .02%)

The data pattern analysis (6/da) of assays 1 - 5 in intensive care unit patients indicates that the majority of discriminant prognostic information is contained in the differential stimulator assays I and II i.e. assays 2,3 while assays 1,4,5 are less informative. Nevertheless it is recommended to perform assays 1,2,3,5 in clinical studies at least in the early phase until reliable data pattern classifications permit the exclusion of certain assays to simplify the panel.


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7.5.6 OXIDATIVE BURST AND PEROXIDASE ACTIVITY:

DCFH-DA: (15/fu, 9/fu, 6/fu, 1/cl) dichlorofluorescin-diacetate MW: 487.3 D,
H2O2 formation and peroxidase activity (low sensitivity and specificity as compared to DHR123)
- DCFH-DA stock solution 4.87mg/ml (10mM) in DMF
- predilute daily 10ul DCFH-DA stock solution with 90ul HBS buffer containing 2mg/ml PI (DCFH-DA 1/10 0.487mg/ml 1mM, PI 2mg/ml 3mM, DMF 10%)

- PMA stock solution: 0.662mg/ml (1.0mM) in DMF, freeze in 5ul portions in 1.5ml Eppendorf conical tubes with snap caps
- predilute freshly with 1ml HBS (PMA 5uM, DMF: 0.5%)
- H2O2: 1/200 (50mM) prediluted from Perhydrol (35% H2O2, Merck) with HBS

assay1: spontaneous oxidation
250ul cell suspension
+5ul DCFH-DA 1/10 0.487mg/ml, PI 2mg/ml in HBS, DMF: 10%
- incubate 15min at 22oC
- measure (DCFH-DA 9.74ug/ml (20uM), PI 40ug/ml (60uM), DMF: 0.2%)

assay2: PMA stimulation (positive control)
250ul cell suspension
+5ul DCFH-DA 1/10 0.487mg/ml, PI 2mg/ml in HBS, DMF: 10%
- incubate 15min at 22oC or 37oC
+5ul PMA 5uM, DMF 0.5% in HBS
- incubate 10min at 22oC or 37oC
- measure (DCFH-DA 9.74ug/ml (20uM), PMA 100nM, PI 40ug/ml (60uM), DMF 0.2%)

assay3: peroxidase activity
250ul cells
+5ul DCFH-DA 1/10 0.487mg/ml, PI 2mg/ml, DMF 10% in HBS
- incubate 15min at 22oC or 37oC
+5ul H2O2 1/200 50mM in HBS from Perhydrol (Merck)
- incubate 10min at 22oC or 37oC
- measure (DCFH-DA 9.74ug/ml (20uM), H2O2 (1mM), PI 40ug/ml (60uM), DMF 0.2%)


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7.5.7 OXIDATIVE BURST:

HE: (16/fu, 15/fu, 9/fu, 1/cl) hydroethidine MW: 315.0 D,
O2- radical formation/general intracellular oxidation,
- HE stock-solution: 3.15mg/ml (10mM) in DMF, freeze at: -180oC, working solution refreeze at: -85oC
- predilute 5ul HE 10mM with 150ul 2mg/ml PI in HBS (HE 1/30, 105ug/ml, 330uM, PI 2mg/ml, 3mM, DMF 3.3%

assay1: spontaneous oxidation
+250ul cell suspension
+5ul HE 1/30 105ug/ml, PI 2mg/ml in HBS
- incubate 15min 37oC
- measure (HE 2.1ug/ml (6.6uM), PI 40ug/ml (60uM), DMF 0.07%)

assay2: PMA stimulation (positive control)

assay3: peroxidase activity
(both assays similarly as above)


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7.5.8 OXIDATIVE BURST:

SIMULTANEOUS O2- and H2O2 formation + intracellular oxidation (DCFH-DA (15/fu, 9/fu, 6/fu, 1/cl) + HE (16/fu, 15/fu, 9/fu), 1/cl)
- predilute: DCFH-DA stock solution 4.87mg/ml (10mM) in DMF 1+9 to 1mM with 2mg/ml PI in HBS daily
- HE stock-solution: 3.15mg/ml (10mM) in DMF, freeze at: -180oC, working solution refreeze at: -85oC
- predilute 5ul HE 10mM with 150ul 2mg/ml PI in HBS (HE 1/30, 105ug/ml, 330uM, PI 2mg/ml, DMF 3.3%)

assay1:
+250ul cell suspension
+5ul DCFH-DA 1/10 0.487mg/ml, PI 2mg/ml, DMF 10% in HBS
+5ul HE 1/30 105ug/ml, PI 2mg/ml, DMF 3.3% in HBS
- incubate 15min 37oC
- measure (DCFH-DA 9.74ug/ml (20uM), HE 2.1ug/ml (6.6uM), PI 40ug/ml (60uM), DMF 0.27%)

assay2: PMA stimulation (positive control)

assay3: peroxidase activity
(both assays similarly as above)


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7.5.9 PROTEASES:
7.5.9.1 R110 Substrate Solutions:

R110: protease substrates
Stock solutions (4mM):
- (Z-Arg2)2-R110 (23/fu, 20/fu, 19/fu, 18/fu, 13/fu, 12/fu, 6/da) cath.B: 6.85mg/ml in DMF, dilute 1/20 with 2mg/ml PI in DMF to 0.274mg/ml (0.2mM)
- (Z-PheArg)2-R110 (23/fu) 20/fu, 19/fu, 18/fu, 13/fu, 12/fu) cath.B: 5.40mg/ml in DMF, dilute 1/20 with 2mg/ml PI in DMF to 0.270mg/ml (0.2mM)
- (Z-Ala2)2-R110 (20/fu, 18/fu, 13/fu, 12/fu, 6/da) cath G: 3.53mg/ml in DMF, dilute 1/20 with 2mg/ml PI in DMF to 0.177mg/ml (0.2mM)
- (NH2-Leu)2-R110 (24/fu, 22/fu, 21/fu) aminopeptidase: 2.22 mg/ml in DMF, dilute 1/20 with 2mg/ml PI in DMF to 0.111 mg/ml (0.2mM)
- (NH2-Phe)2-R110 24/fu, 22/fu, 21/fu) aminopeptidase: 2.49 mg/ml in DMF, dilute 1/20 with 2mg/ml PI in DMF to 0.125 mg/ml (.2mM)


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7.5.9.2 PROTEASE INHIBITOR SOLUTIONS

- DFP: 1M stock solution in DMSO, store at -20oC, dilute 1+4 with HBS prior to use, observe manufacturers safety instruction and handle DFP with extreme care because it is a potent neurotoxin (volatile cholinesterase inhibitor, antidot: atropine, detoxification in 5M NaOH, prepare DFP stock solution and 1+4 dilution under ventilated hood, DFP is stable for several days in protein free physiological solutions !)
- Z-Phe-Ala-CHN2, 3.94mg/ml (10mM) stock solution in DMSO, store at -20oC, dilute 1+4 with HBS prior to use (2mM).
- Z-Leu-CHN2, 2.00mg/ml (10mM) stock solution in DMSO, store at -20oC, dilute 1+49 with BS prior to use (0.2mM).


7.5.9.3 PROTEASE ACTIVITY ASSAYS

250ul cell suspension 1x106-1x107 cells/ml
+5ul substrate 0.2mM in DMF
- incubate 15-30min at 22oC or 37oC
+5ul PI 2mg/ml in HBS
- incubate 5min at 22oC
- measure (R110 substrate 4uM, PI 40ug/ml (60uM))


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7.5.9.4 PROTEASE INHIBITION ASSAYS:

cathepsin B,H,L inhibitory assay (cystein proteinases):
250ul cell suspension 1x106-1x107 cells/ml
+5ul Z-Phe-Ala-CHN2 2mM in HBS
- incubate 15min at 22oC or 37oC
+5ul substrate 0.2mM in DMF
- incubate 15-30min at 22oC or 37oC
+5ul PI 2mg/ml in HBS
- incubate 5min at 22oC
- measure (R110 substrate 4uM, Z-Phe-Ala-CHN2 40uM, PI 40ug/ml (60uM))

cathepsin G inhibitory assay (serine proteinases):
250ul cell suspension 1x106-1x107 cells/ml
+5ul DFP 50mM in HBS
- incubate 15min at 22oC or 37oC
+5ul substrate 0.2mM in DMF
- incubate 15-30min at 22oC or 37oC
+5ul PI 2mg/ml in HBS
- incubate 5min at 22oC
- measure (R110 substrate 4uM, DFP 1mM, PI 40ug/ml (60uM))

aminopeptidase inhibitory assay (metallo proteinases):
250ul cell suspension
+5ul Leu-CMK (chloromethylketone) inhibitor 0.2mM in HBS
- incubate 15min at 20oC or 37oC
+5ul substrate 0.2mM
- incubate 15min at 20oC or 37oC
+5ul PI 2mg/ml in DMF
- incubate 5min at 22oC
- measure (R110 substrate 4uM, CMK 40mM, PI 40ug/ml (60uM))

R110: reaction product control assay (control, only once to setup flow cytometer):
- prepare R110 stock solution 7.33mg/ml (20mM) in HBS
- predilute 1/100 with HBS (R110 0.2mM)
250ul cells suspension 1x106-1x107 cells/ml
+5ul R110 0.2mM (73.3ug/ml) in HBS
- incubate 15-30min at 22oC or 37oC
+1000ul HBS, centrifuge 30 seconds, 1000 x g
+250ul HBS to resuspend sediment
+5ul PI 2 mg/ml in HBS
- incubate 5 min at 22oC
- measure (R110 1.46ug/ml (4uM), PI 40ug/ml (60uM))


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7.5.9.5 R110 PROTEASE SUBSTRATE STORAGE:

R110 protease substrate storage and aliquotation:
- take 250ul 4mM substrate solution in DMF which is good for the staining of 1000 cell samples
- aliquot 50ul portions into five Eppendorf cap or screw top plastic vials
- store four aliquots at -85oC

aliquots:
- take the remaining 50ul aliquot:
50ul substrate 4mM
950ul DMF
1000ul substrate solution 0.2mM for staining of 200 samples
- aliquot this 0.2mM substrate solution in 50ul portions into Eppendorf cap or screw top vials and store at -85oC
- thaw for measurements one 0.2mM aliquot which is good for the staining of 10 cell samples. Left over substrate solution may be refrozen or stored at 4oC in the dark in a refrigerator.


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7.5.10 Esterase activity:

FDA: esterase substrate fluorescein diacetate MW: 416 D
- predilute: FDA stock solution 4.16mg/ml (10mM) in DMF daily 1/100 with 2mg/ml PI in HBS (FDA 1/100 41.6ug/ml 100uM)

assay:
250ul cell suspension
+5ul FDA 41.6ug/ml, PI 2mg/ml in HBS
- incubate 15 min 22oC
- measure (FDA 0.83ug/ml (2uM), PI 40ug/ml (60uM))


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7.5.11 DNA/RNA:

AO: (7/fu, 3/fu, 2/cl, 1/cl) vital DNA/RNA acridine orange MW: 301.83 D

assay:
250ul cell suspension
+5ul AO 0.4mg/ml, PI 2mg/ml in DMF
- incubate 15 min 0oC or 22oC
- measure (AO 8ug/ml (26uM), PI 40ug/ml (60uM))


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8.CELL PREPARATION AND FIXATION

8.1 Tissue Cells

Cells from tissues may be obtained mechanically or enzymatically. Enzymatic treatment requires usually 37oC incubation which may modify or remove cell surface antigens as well as alter the metabolic status of viable cells as a consequence of enzyme action.

Tissues are removed from the body, suspensed and short term stored in buffered saline or tissue culture solution at 0-4oC on ice. Small tissue pieces (50-100mg) are cut with a blade knife (no scissors because of tissue compression) and chopped with a multiblade tissue chopper (e.g. McIlwain) in two steps with 90o rotated sample stage.

The chopped tissue is resuspended in 2-4ml physiological solution in a 50ml Falcon tube with conical bottom, mechanically treated by 20-30 time sucking back and forth with plastic tip pipette (e.g. Eppendorf) whose tip has been cut to provide an opening of 1.5-2.5mm. Bubble formation is carefully avoided. Cells are washed twice by centrifugation at 200xg for 10min with 50ml physiological solution. Cells are carefully resuspended at each centrifugation step in 3-5ml physiological solution by sucking the sediment several times back and forth without bubbles with the plastic tip pipette before filling up to 50ml. Following the last centrifugation, the resuspended cells are ready for viable cell assays or fixation. All working steps and centrifugations are done at 0-4oC to maintain the metabolic state of the cells as good as possible.


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8.2 Leukocytes and Bone Marrow
8.2.1 Erythrocyte Lysis

Blood or bone marrow cell samples for flow cytometric immunophenotyping are obtained by erythrocyte lysis following suspension of cells in 0.9% ammonium chloride (NH4Cl) solution pH 7.35. Since substantial acidification followed by alkalinization occurs in unlyzed leukocytes, the lyzing solutions for immunophenotyping are *unsuitable* for flow cytometric cell function studies.


8.2.2 Cell Sedimentation
8.2.2.1 Histopaque 1g Sedimentation

3ml of a Histopaque 1.077g/ml solution (e.g. Sigma 1077-1) are filled into a 10ml polycarbonate or glass tube. 3 ml heparin anticoagulated blood (10-20 IU/ml) are layered directly from the syringe by running the blood via the wall of the about 30o inclined tube onto the Histopaque. Bone marrow samples are filled into a separate test tube and sucked back and forth without bubbles using a plastic tip pipette as described under 8.1 Heparin is preferred to EDTA or citrate to maintain normal Ca2+ level in the blood plasma. This is essential for proper cell function.

Erythrocytes aggregate heavily at the blood/dextran interface and quickly sediment into the Histopaque while non aggregating and slower sedimenting leuko- and thrombocytes remain in their natural environment. After about 30-45min at room temperature most erythrocytes have sedimented while the upper 2ml of the suspension containg leuko- and thrombocytes are carefully removed with a 1ml plastic tip pipette and stored at 0-4oC on ice until staining. Cell are >90% leuko- and thrombocytes.


8.2.2.2 1g Sedimentation

Instead of Histopaque underlayering, 10ml heparinized blood can also be allowed to sediment for 1 or 2h at room temperature in a narrow i.e. 1-1.5cm diameter polycarbonate or glass test tube. The upper 1ml is carefully removed with a 1ml plastic tip pipette. The advantage is that no centrifugation is required. More blood and a longer sedimentation time are, however, required. The leukocyte enrichment factor is also substantially lower than in 8.2.2.1 as well as in 8.2.2.3.


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8.2.2.3 Centrifugation

Heparinized blood is centrifuged for 5min at 150-200g. The buffy coat, barely layered on top of the still loose erythrocyte column is carefully removed. This procedure provides about 10-50 fold leukocyte enriched erythrocyte suspensions in plasma. This is a good environment e.g. for phagocytosis assays (14/fu, 10/fu, 9/fu, 7/fu, 6/fu, 2/cl, 1/cl) because leuko- and thrombocyte aggregation by the presence of bacteria is largely inhibited.


8.2.2.4 Bacteria Phagocytosis

(14/fu, 10/fu, 9/fu, 7/fu, 6/fu, 2/cl, 1/cl) 50ul leukocyte preparation (buffy coat after 200g centrifugation) in 1.5ml Eppendorf cups
+5ul vital E-coli K12 strain (Sigma) bacteria suspension (3x10**9/ml) photometric extinction 0.100 at 350nm for 3x10**7 bacteria/ml in HBS-buffer
- keep 10ul of the assay at 0oC and dilute with 1ml cold HBS-buffer - incubate the remaining 40 ul in water bath at 37oC, mix gently every 15min
- control assays are 50ul leukocyte preparation with 5ul HBS-buffer, incubated and treated as the assays with bacteria. - remove 10ul aliquots at 30 and 90min and dilute with 1ml cold HBS-buffer, store all samples at 0-4oC on ice. - stain 250ul diluted samples (ca 1x10**7 cells/ml) with any of the above functional stains in the presence of PI.


8.3 Cell Fixation

Cell fixation preserves cells for longer storage periods. It is important for flow cytometry to fix in suspension, in physiological solutions without protein and at 0-4oC to maintain the cells as close as possible to their actual metabolic state.


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8.3.1 Aldehydes

Aldehydes fix by denaturation and chemical modification of proteins i.e. by covalent reaction with free amino groups of e.g. lysine residues. The fixation frequently alters peptide chain antigens of intracellular proteins while the glyco-antigens of the cell membrane glycocalix remain largely unaffected. Cell become rigid because protein cross linking occurs i.e. they suspend well. The cell membranes of intact cells remain relatively unpenetrable to larger molecules e.g. antibodies. Electrical cell volume sizing remains largely unaffected but DNA stainability is substantially reduced. This may be overcome by longer staining times.

Formaldehyde or paraformaldehyde are mostly used in flow cytometry to avoid the UV or 488nm excited cellular autofluorescence in the blue and green induced by the well known cross-linking properties of glutaraldehyde.

a. Freshly prepared (weekly) 3.5% formaldehyde solution:
1 part 35% formaldehyde (formalin) solution (Merck 4003, MW:30.03)
9 parts HBS-buffer pH 7.35
+ ... ml 0.1N NaOH (MW: 40.0) until pH 7.35 remains stable
discard when pH <7.0

b. Fixation:
1 part protein free cell suspension (5x10**6 - 5x10**7 cells/ml) in HBS-buffer pH 7.35
+1 part fixation solution (3.5% formaldehyde)
- mix rapidly by shaking of inject fixative quickly into homogeneous cell suspension e.g. by 5ml plastic tip pipette
- fix for at least 1h
- keep fixed cells at 0-4oC in the dark in the fixative
- wash cells twice in 50ml HBS-buffer prior to use e.g. for antibody incubation for removal of fixative.


8.3.2 Organic solvents

Methanol, ethanol, acetone denature protein mainly by removal of bound H2O molecules but without covalent modifications of protein structure. Organic fixative remove furthermore membrane and structural lipids to some extent i.e. the cells become permeable e.g. to antibody molecules. Due to shrinking by water removal and permeabilization, the electrical volume sizing signals as well as light scatter signals are substantially modified as compared to viable cells. Intracellular as well as cell membrane antigens are usually well preserved in organic fixatives and DNA staining is quick and with comparatively good CV's.

Problems arise from cell clumping by ethanol or acetone during or after fixation. Methanol at 0-4oC is a well performing fixative for blood, bone marrow, smear or mechanically obtained tissue cell suspensions.

a. Smear and tissue cells:
3ml protein free cell suspension (5x10**6 - 5x10**7 cells/ml)
+ 7ml (Merck Nr.6009 p.a)
- mix rapidly or inject 0-4oC methanol
- fix for at least 1h at 0-4oC
- centrifuge 10min at 200g, remove supernatant e.g. with Pasteur pipette until 1.5-2ml
- resuspend samples with plastic tip pipette
- store samples at 0-4oC in the cold in 3ml glass vials with screw cap
- wash cells twice with 50ml HBS-buffer prior to use e.g. antibody incubation

b.Strongly erythrocyte containing cell suspension:
1 part protein free cell suspension as under a.
+1 part methanol
- mix rapidly or inject methanol
- wait ca 5min at 0oC to lyse erythrocytes
- centrifuge for 5-10min at 200g to sediment unlysed cells
- remove supernatant immediately by suction
- resuspend sediment with 2ml 70% methanol (7 parts methanol, 3 parts HBS-buffer ph 7.35) by plastic tip pipette
- store samples at 0-4oC in the cold in 3ml glass vials with screw cap
- wash cells twice with 50ml HBS-buffer prior to use e.g. antibody incubation


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9. SELECTED LITERATURE REFERENCES (> further references )

9.1 Flow Cytometric Cell Function Assays:

25. Amann S, Reinke C , Valet G, Moser U, Leuenberger H. Flow cytometric investigation of cellular metabolism during oxidative stress and the effect of tocopherol. Internat J Vit Nutr Res 69:356-361(1999)
24. Ganesh S, Klingel S, Kahle H, Valet G. Flow cytometric determination of aminopeptidase activities in viable cells using fluorogenic rhodamine110 substrates. Cytometry 20:334-340(1995) *external link (PDF).
23. Elsherif T, Kahle H, Klingel S, Ganesh S, Valet G. Early functional changes in human T-cells induced to apoptosis by X-irradiation or cortisone. Cytometry Suppl.7 p 25(1994)
22. Klingel S, Ganesh S, Kahle H, Valet G. Flow cytometric aminopeptidase and cathepsin D determination in living cells by fluorogenic rhodamine substrates. Cytometry Suppl.7 p 77(1994)
21. Klingel S, Ganesh S, Kahle H, Valet G. Fluorogenic rhodamine110 substrates for the flow cytometric determination of aminopeptidase and cathepsin D activities in living cells. Anal.Cell.Pathology 6:257(1994)
20. Klingel S, Rothe G, Kellermann W, Valet G. Flow cytometric determination of cysteine and serine proteinase activities in living cells with rhodamine110 substrates. Methods Cell Biology 41:449-459(1994)
19. Banati RB, Rothe G, Valet G, Kreutzberg GW. Detection of lysosomal cysteine proteinases in microglia: Flow cytometric measurement and histochemical localization of cathepsin B and L. Glia 7:183-191(1993)
18. Rothe G, Valet G. Measurement of mononuclear phagocytic cathepsin B/L activity with (N- benzyloxycarbonyl-Arg-Arg)2-rhodamine110, In: Handbook of Flow Cytometric Methods, Eds: Robinson JP, Darzynkiewicz Z, Dean P, Dressler L, Tanke H, Wheeless L, Wiley-Liss Inc, New York 1993, p 202-203
17. Rothe G, Valet G. Measurement of neutrophil elastase activity with (N-benzyloxycarbonyl-Ala-Ala)2-rho- damine110, In: Handbook of Flow Cytometric Methods, Eds: Robinson JP, Darzynkiewicz Z, Dean P, Dressler L, Tanke H, Wheeless L, Wiley-Liss Inc, New York 1993, p 200-201
16. Rothe G, Valet G. Measurement of NADPH oxidase activity with hydroethidine, In: Handbook of Flow Cytometric Methods, Eds: Robinson JP, Darzynkiewicz Z, Dean P, Dressler L, Tanke H, Wheeless L, Wiley-Liss Inc, New York 1993, p 159-160
15. Rothe G, Valet G. Simultaneous measurement of NADPH oxidase activity and phagosomal oxidation with hydroethidine and 2'7'-dichlororfluorescin diacetate, In: Handbook of Flow Cytometric Methods, Eds: Robinson JP, Darzynkiewicz Z, Dean P, Dressler L, Tanke H, Wheeless L, Wiley-Liss Inc, New York 1993, p 157-158
14. Rothe G, Valet G. Measurement of phagosomal hydrogen peroxide production with dihydrorhodamin123 In: Handbook of Flow Cytometric Methods, Eds: Robinson JP, Darzynkiewicz Z, Dean P, Dressler L, Tanke H, Wheeless L, Wiley-Liss Inc, New York 1993, p 155-156
13. Assfalg-Machleidt I, Rothe G, Klingel S, Banati R, Mangel WF, Valet G, Machleidt W. Membrane permeable fluorogenic rhodamine substrates for selective determination of cathepsin L. Biol Chem Hoppe Seyler 373:433-440(1992)
12. Rothe G, Klingel S, Assfalg-Machleidt I, Machleidt W, Zirkelbach C, Banati R, Mangel WF, Valet G. Flow cytometric analysis of protease activities in vital cells. Biol Chem Hoppe Seyler 373:547-554(1992)
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11. Banati RB, Rothe G, Valet G, Kreutzberg GW. Respiratory burst in brain macrophages: A flow cytometric study on cultured rat macrophages. Neuropath Appl Neurobiol 17:223-230(1991)
10. Rothe G, Emmendörfer A, Oser A, Roesler J, Valet G. Flow cytometric measurement of the respiratory burst activity of phagocytes using dihydrorhodamine 123. J Immunol Methods 138:133-135(1991)
9. Rothe G, Valet G. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2'7'-dichlorofluorescin. J Leuk Biol 47:440-448(1990)
8. Rothe G, Oser A, Valet G. Dihydrorhodamine123: a new flow cytometric indicator for respiratory burst activity in neutrophil granulocytes. Naturwiss 75:354-355(1988)
7. Rothe G, Valet G. Phagocytosis, intracellular pH, and cell volume in the multifunctional analysis of granulocytes by flow-cytometry. Cytometry 9:316-324(1988) *external link (PDF).
6. Burow S, Valet G. Flow-cytometric characterization of stimulation, free radical formation, peroxidase activity and phagocytosis of human granulocytes with 2,7-dichlorofluorescin (DCF). Eur J Cell Biology 43:128-133(1987)
5. Treumer J, Valet G. Flow-cytometric determination of glutathione alterations in vital cells by o- phthaldialdehyde (OPT) staining. Exp Cell Res 163:518-524(1986)
4. Valet G, Raffael A. Determination of intracellular calcium in vital cells by flow- cytometry. Naturwiss 72:600-602(1985)
3. Valet G. A new method for fast blood cell counting and partial differentiation by flow-cytometry Blut 49:83-90(1984)
2. Valet G, Raffael A. Cyto-P-Check, Determination of intracellular pH and esterase activity in vital cells by flow-cytometry. Paesel, Frankfurt 1984 p 1-21
1. Valet G, Raffael A, Moroder L, Wünsch E, Ruhenstroth-Bauer G. Fast intracellular pH determination in single cells by flow- cytometry. Naturwissenschaften 68:265-266(1981)
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9.2 Cytostatic Drug Assays:

9. Wulf G, Falk H, Weiershausen U, Valet G. Antimetabolic and cytostatic potential of 4-amino-N-(2'-aminophenyl)-benzamide (Dinaline) on adriamycin-sensitive (FL) and resistant (ARN) Friend leukemia cells. J Cell Pharmacol 1:109-118(1990)
8. Lampidis TJ, Savaraj N, Valet GK, Tevorrow K, Fourcade A, Tapiero H. Relationship of chemical charge of anticancer agents to increased accumulation and cytotoxicity in cardiac and tumor cells: relevance to multidrug resistance. J Cellular Pharmacology 1:16-22(1989)
7. Lampidis TJ, Castello C, Giglio A, Pressman BC, Viallet P, Trevorrow KW, Valet GK, Tapiero H, Savaraj N. Relevance of the chemical charge of rhodamine dyes to multiple drug resistance. Biochem Pharmacol 38:4267-4271(1989)
6. Hasman M, Valet G, Tapiero H, Trevorrow K, Lampidis T. Membrane potential differences between adriamycin sensitive and resistant cells as measured by flow cytometry Biochem Pharmacol 38:305-312(1989)
5. Neubauer A, Valet G, Huhn D. Flow-cytometric determination of intracellular pH, esterase activity and cell volume in human leukemic cell lines following in-vitro incubation with cytostatic drugs. Anal Cell Pathology 2:49-58(1989)
4. Neubauer A, Sauer R, Valet G. Cytostatic drug testing in human leukemias by multiparameter flow-cytometry. Blut 55:433-445(1987)
3. Valet G, Warnecke HH, Kahle H. New possibilities of cytostatic drug testing on patient tumor cells by flow cytometry. Blut 49:37-43(1984)
2. Valet G, Warnecke HH, Kahle H. Cyto-P-Check, Cytostatic drug testing by flow-cytometry. Paesel, Frankfurt 1984 p 22-30
1. Wirsching R, Ruessmann L, Koller J, Valet G. Zytostatika-Testung: Ein neuartiges Verfahren zur Beurteilung der Wirkung auf menschliche Tumorzellen. Münchn Med Wschr 125:31(1983)

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9.3 Medical Bioinformatics ( Data Pattern Classification):

9. Valet G. Prädiktivmedizin mittels Zytomik: Möglichkeiten der Datenmusteranalyse. In: Klinische Durchflusszytometrie. Eds: U Sack, A Tarnok, G Rothe. Karger Verlag, Basel (2007) p 237-250
8. 180. Valet G. Human cytome project: A new potential for drug discovery. In: Las Omicas genomica, poteomica, citomica y metabolomica: modernas tecnologias para desarrollo de farmacos. Ed: Real Academia Nacional de Farmacia, Madrid 2005 p 207-228
7. Valet G, Kahle H, Otto F, Bräutigam E, Kestens L. Prediction and precise diagnosis of diseases by data pattern analysis in multiparameter flow cytometry: Melanoma, Juvenile Asthma, HIV-infection. In: Cytometry (3rd edition), Eds: Darzynkiewicz Z, Robinson JP, Crissman HA, Academic Press, San Diego. Methods in Cell Biology 64:487-508(2001)
6. Valet G, Roth G, Kellermann W. Risk assessment for intensive care patients by automated classification of flow cytometric oxidative burst, serine and cysteine proteinase activity measurements using CLASSIF1 triple matrix analysis. In: Cytometric Cellular Analysis, Eds: Robinson JP, Babcock G, Wiley-Liss, New York 1998, p 289-306
5. Valet G, Höffkes HG. Automated classification of patients with chronic lymphatic leukemia and immunocytoma from flow cytometric three colour immunophenotypes. Cytometry (CCC) 30:275-288(1997) *external link (PDF).
4. Valet G, Valet M, Tschöpe D, Gabriel H, Rothe G, Kellermann W, Kahle H. White cell and thrombocyte disorders: Standardized, self-learning flow cytometric list mode data classification with the CLASSIF1 program system. Ann NY Acad Sci 677:233-251(1993)
3. Valet G, Warnecke HH, Kahle H. Automated diagnosis of malignant and other abnormal cells by flow-cytometry using the DIAGNOS1 program system. In: Clinical Cytometry and Histometry, Eds: Burger G, Ploem J, Goerttler K, Academic press, London 1987, p 58-65
2. Valet G, Rüssmann L, Wirsching R. Automated flow-cytometric identification of colo-rectal tumor cells by simultaneouus DNA, CEA-antibody and cell volume measurements. J Clin Chem Clin Biochem 22:935-942(1984)
1. Valet G, Ormerod MG, Warnecke HH, Benker G, Ruhenstroth-Bauer G. Sensitive three-parameter flow-cytometric detection of abnormal cells in human cervical cancers: A pilot study. J Canc Res Clin Oncol 102:177-184(1981)
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9.4 Human Cytome Project, Medical Cytomics, Clinical Cytomics:

32. Valet G, Murpy RF, Robinson JP, Tarnok A, Kriete A. Cytomics - from cell states to predictive medicine. In: Computational Systems Biology. Eds: Kriete A, Eils R., Elsevier, Amsterdam 2006 p 363-381
31. Valet G. Cytomics, human cytome project and systems biology: top-down resolution of the biomolecular complexity of organisms by single cell analysis. Cell Proliferation 38:171-174(2005)
30. Valet G. Human cytome project: A new potential for drug discovery. In: Las Omicas genomica, proteomica, citomica y metabolomica: modernas tecnologias para el desarrollo de farmacos. Ed: Real Academia Nacional des Farmacia, Madrid 2005 p 207-228
29. Valet G. Human cytome project, cytomics and systems biology: the incentive for new horizons in cytometry. Cytometry 64A:1-2(2005) *external link (PDF).
28. Valet G. Cytomics: an entry to biomedical cell systems biology. Cytometry 63A:67-68(2005) *external link (PDF).
27. Valet G. Diagnostic versus predictive flow cytometry. Lab Hematol 10:166-168(2004)
26. Valet G, Tarnok A. Potential and challenges of a human cytome project. JBRHA 18:87-91(2004)
25. Valet G, Tarnok A. Cytomics - New technologies: Towards a human cytome project. Cytometry 59A:167-171(2004) *external link (PDF).
24. Valet G, Hoeffkes HG. Data pattern analysis for the individualized pretherapeutic identification of high risk diffuse large B-cell lymphoma (DLBCL) patients by cytomics. Cytometry 59A:232-236(2004) (*external link PDF).
23. Greve B, Valet G, Humpe A, Tonn T, Cassens U. Flow cytometry in transfusion medicine: Development, strategies and applications. Transf Med Hemother 31:152-161(2004)
22. Tarnok A, Valet GK. Cytomics in predictive medicine. In: Advanced Biomedical and Clinical Diagnostic Systems II. Eds: GE Cohn, WS Grundfest, DA Benaron, T Vo-Dinh, Proceedings SPIE, Bellingham, WA 2004, Vol 5318, p 12-22
21. Valet G, Tarnok A. Cytomics in predictive Medicine In: Business Briefing: Future Drug Discovery 2004 Ed: E Cooper, World Markets Research Center Ltd, London (2004) p 1-3. (*external link PDF)
20. Valet G. Predictive medicine by cytomics and the challenges of a human cytome project. In: Business Briefing: Future Drug Discovery 2004, Ed: E Cooper, World Markets Research Center Ltd, London (2004) p 46-51. (*external link PDF)
19. Valet G, Cornelissen J, Lamers G, Gratama J. Predictive medicine by cytomics: identification of high risk patients in bone marrow stem cell transplantation. Cytometry 53B:62-63(2003)
18. Valet G, Repp R, Link H, Ehninger G, Gramatzki M and SHG-AML study group. Pretherapeutic identification of high risk acute myeloid leukemia (AML) patients from immunophenotype, cytogenetic and clinical parameters. Cytometry 53B:4-10(2003) *external link (PDF).
17. Valet G, Tarnok A. Cytomics and Predictive Medicine Cytometry 53B:1-3(2003) *external link (PDF).
16. Jacobi AM, Odendahl M, Reiter K, Bruns A, Burmester GM, Radbruch A, Valet G, Lipsky PE, Dörner T. Correlation between circulating CD27high plasma cells and disease activity in patients with systemic lupus erythematosus. Arthritis & Rheumatism 48:1332-1342(2003)
15. Valet G. Predictive Medicine by Cytomics: Potential and Challenges. J.Biol.Regulators 16:164-167(2002)
14. Valet G, Arland M, Franke A, Kahl C, Höffkes HG . Discrimination of chronic lymphocytic leukemia of B-cell type by computerized 3-color flow cytometric immunophenotypes of bone marrow aspirates and peripheral blood. Lab.Hematology 8:134-142(2002)
13. Bocsi J, Hambsch J, Osmancik P, Schneider P, Valet G, Tarnok A. Preoperative prediction of pediatric patients with effusions and edema following cardiopulmonary bypass surgery by serological and routine laboratory data. Critical Care 6:226-233,(2002)
12. Tarnok A, Bocsi J, Pipek M, Osmancik P, Valet G. Preoperative prediction of postoperative edema and effusion in pediatric cardiac surgery by altered antigen expression patterns on granulocytes and monocytes. Cytometry (CCC) 46:247-253(2001) *external link (PDF).
11. Bartsch R, Arland M, Lange S, Kahl C, Valet G, Höffkes HG. Lymphoma discrimination by computerized triple matrix analysis of list mode data from three-color flow cytometric immunophenotypes of bone marrow aspirates. Cytometry 41:9-18(2000) *external link (PDF).
10. van Driel BEM, Valet GK, Lyon H, Hansen U, Song JY, van Noorden CJF Prognostic estimation of survival of colorectal cancer patients with the quantitative histochemical assay of G6PDH activity and the multiparameter classification program CLASSIF1. Cytometry(Comm.Clin.Cytometry) 38:176-183(1999) *external link (PDF).
9. Tarnok A, Pipek M, Valet G, Richter J, Hambsch J, Schneider P. Children with posty-capillary leak syndrome can be distinguished by antigen expression on neutrophil and monocytes. in: System and Technologies for Clinical Diagnostics and Drug Discovery II, eds: Cohn GE, Owicki JC, Katzir A. SPIE Vol.3603:61-71(1999)
8. Tarnok A, Hambsch J, Borte M, Valet G, Schneider P. Immunological and serological discrimination of children with and without post-surgical capillary leak syndrome. in: The Immune Consequences of Trauma, Shock and Sepsis Vol.III, Ed: E.Faist, Monduzzi Editore, Bologna 1997, p 845-849
7. Höffkes HG, Schmidtke G, Schmücker U, Brittinger G, Valet G. Computerized analysis of cells from patients with acute myelogeneous leukemia prepared by density gradient centrifugation or erythrocyte lysis and measured by flow cytometry. Lab.Hematol.1:128-134(1995)
6. Rothe G, Kellermann W, Briegel J, Schaerer B, Valet G. Activation of neutrophils by tumor necrosis factor-a during sepsis. in: Immune Consequences of Trauma, Shock and Sepsis Vol.II, Ed: E Faist, J Ninnemann, D Green, Springer Verlag, Berlin 1993, p 727-733
5. Liewald F, Hatz R, Storck M, Orend KH, Weiss M, Wulf G, Valet G, Sunder-Plasmann L. Prognostic value of deoxyribonucleic acid aneuploidy in primary non small-cell lung carcinomas and their metastases. J.Thoracic Cardiovasc.Surgery 105:1476-1482(1992)
4. Liewald F, Sunder-Plassmann L, Dienemann H, Kahle H, Wulf G, Valet G. Prognostic value of flow cytometrically determined DNA-ploidy, intracellular pH and esterase activity of non-small cell lung carcinoma. Anal Cell Pathology 4:103-114(1992)
3. Liewald F, Demmel N, Wirsching R, Kahle H, Valet G. Intracellular pH, esterase activity and DNA measurements of human lung carcinomas by flow-cytometry Cytometry 11:341-348(1990) *external link (PDF).
2. Rothe G, Kellermann W, Valet G. Flow cytometric parameters of neutrophil function as early indicators of sepsis- or trauma-related pulmonary or cardiovascular organ failure. J Lab Clin Invest. 115:52-61(1990)
1. Rothe G, Kellermann W, Valet G. Flow cytometric analysis of phagocytosis, respiratory burst, intracellular pH and cytosolic free calcium of granulocytes of posttraumatic and septic patients. in: Immune Consequences of Trauma, Shock and Sepsis, eds: E Faist, J Ninnemann, D Green, Springer Verlag, Berlin 1989, p 235- 240
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9.5 Marine Plankton:

1. Sieracki M, Valet G, Cucci T. Report on advanced workshop on fluorescent probes for marine flow cytometry: Use of fluorescent probes in the study of phytoplankton physiology and cellular biochemistry. Signal and Noise 6:1-2(1993)

9.6 General Flow Cytometry:


4. Valet G. Concepts & developments in flow cytometry & cytomics at Max-Planck-Institut für Biochemie, Martinsried (1960-2006). In: ed JP Robinson. 60 Years Innovation in Cytometry (Purdue CD10) ISBN 978-1-890473-10-5 (2007)
3. Valet G. Die Durchflusszytometrie im Wandel der Forschungskonzepte. In: Angewandte Durchflusszytometrie. Eds: U Sack, A Tarnok, G Rothe. Karger Verlag, Basel (2007) p 1-26
2. Valet G. Past and present concepts in flow cytometry: A European perspective. JBRHA 17:213-222(2003)
1. Raffael A, Nebe CT, Valet G. Grundlagen der Durchflußzytometrie. In: Durchflußzytometrie in der klinischen Zelldiagnostik, Eds: Schmitz G, Rothe G, Schattauer Verlag, Stuttgart 1994, p 3-49.
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