FUNCTIONAL CELL BIOCHEMISTRY BY FLOW CYTOMETRY

G.Valet

1. Cell Structure and Cell Function

1.1 Structural flow cytometry:

 The flow cytometric determination of structural cellular constituents (Tab.1) is in use for many years e.g. for the determination of DNA distributions, cellular antigen contents or natural pigment analysis like chlorophyll or phycobiliproteins in plankton cells. Simultaneous biophysical measurements of electrical or optical (FSC) cell volume and optical granularity (SSC) complement the cell characterization.

1.2 Functional flow cytometry:

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

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 for a moment a cell with a house: when somebody puts on his coat, no structural but a functional change has occured because neither the number of persons nor the coats change. Recognition of this action provides the diagnosis of the functional change but potentially more important the prognosis that somebody will soon leave the house.

The extraction of prognostic features from cell function measurements is of high interest for medicine but also for cell biological research in general.

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2. Cell function:

2.1 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 and digital imaging techniques has really opened the large potential for cell function measurement with fluorescent dyes.

The main advantage of the microscope is the possibility for detailed morphological single cell observation. Disadvantages are difficulty of quantitation, comparatively low speed of analysis, sample exposure to high light intensities causing fluorescence bleaching, sample heating and observation of cells in the very small fluid volume under the cover slip. The prolonged exposure to high light intensities may influence the biochemical behaviour of cells.

2.2 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. This outweighs the usual impossibi lity to investigate the same cell several times which, in contrast, is very easy under 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. In most instances the stained cell sample can then be measured immediately. In some instances there is a centrifugation step followed by resuspension of the cells in buffer 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 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.

3. Flow cytometer setup for functional stains:

3.1 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 transmembrane potential, rhodamine123 (R123) mitochondrial potential, dihydrorhodamine123 (DHR) or dichlorofluorescin (DCFH) metabolic burst measurements, rhodamine110 (R110) 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) for intracellular pH- measurements, INDO1 calcium stain or orthophtaldialdehyde (OPT) free gluta thione 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.

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4. 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.

5. Cytometry on the INTERNET:

Information on new developments can be obtained on Internet from the CYTORELAY node within the www-net. The address (URL) is: http://www.biochem.mpg.de/valet/cytorel.html

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6. FUNCTIONAL CELL STAINS FOR FLOW-CYTOMETRY

6.1 DYES:
ADB = 1,4-diacetoxy-2.3-dicyano-benzene (MW: 244.2) Nr.50-010-002, Nr.1383 205 Boehringer, D-68298 Mannheim, Nr.266707 Calbiochem, D-65812 Bad Soden, Germany
INDO1/AM = INDO1 pentaacetoxymethyl-ester (MW: 1009.9), Nr.402096, Calbiochem, D-65812 Bad Soden, Nr.1081 217 Boehringer, D-68298 Mannheim, Germany
OPT = ortho-phthaldialdehyde (MW: 134.1), Nr.P-1378, Sigma, St.Louis, USA
HOECHST 33258 = Bisbenzimide H33258 (MW: 623.97), Serva Nr.15090, D-69115 Heidelberg, Germany, Nr.B-2883 Sigma, St.Louis, USA
HOECHST 33342 = Bisbenzimide H33342 (MW: 615.99), Serva Nr.15091, D-69115 Heidelberg, Germany, Nr.B-2261 Sigma, St.Louis, USA
DIOC6 = 3,3-dihexyl-oxacarbocyanin (MW: 572.53), Nr.14414, Eastman Kodak, Rochester, New York, USA
R123 = rhodamine 123 (MW: 380.8), Nr.R-8004, Sigma, St.Louis, USA
DCFH-DA = 2',7'-dichlorofluorescin-diacetate, (MW: 487.3), Nr.19353, Serva, D-69115 Heidelberg, Germany
DCF = 2',7'-dichlorofluorescein (MW: 401.2), Nr.19524, Serva, D-69115 Heidelberg, Germany
DHR123 = dihydrorhodamine 123 (MW: 346), Nr.D-632, Molecular Probes, Eugene, Oregon, USA, Belgium: Becton Dickinson European HQ, Denderstraat 24, B-9320 Erembodegem-Aalst, Tel: +32/53/720211, Fax: +32/53/720450
HE = hydroethidine (MW: 315) , Nr.17084, Polysciences, Warrington, PA, USA, Germany: Handelsstr.3, Postfach 1130, D-69208 Eppelheim, Tel: 06221/765767, Fax: 06221/764620
SNARF1 = carboxy SNARF1 AM acetate, (MW: 568 Nr.C-1271, Molecular Probes, Inc., Eugene, OR 97402-0414, USA
FLUO3/AM = FLUO-3 AM cell permeant, (MW: 1130), Nr.F-1241, Molecular Probes, Inc., Eugene, OR 97402-0414, USA
R110 = rhodamine 110 (MW: 366.8), Nr.136 1468, Eastman Kodak, Rochester, New York, USA, or: Exciton Chemical Co, Inc, Dayton, Ohio, USA
(Z-Ala2)2-R110 = cathepsin G serine protease substrate (MW: 882.94), Nr.R-6504, Molecular Probes, Inc., Eugene, OR 97402-0414, USA
(Z-Arg2)2-R110 = cathepsin B,L cysteine proteinase substrate (MW: 1369.22), laboratory synthesis
(Z-PheArg)2-R110 = cathepsin B,(L) cysteine proteinase substrate (MW: 1351.18), Nr.R-6502, Molecular Probes, Inc., Eugene, OR 97402-0414, USA
(NH2-Leu)2-R110 = aminopeptidase metalloproteinase substrate (MW: 556.7) , Nr.R-6509, Molecular Probes, Inc., Eugene, OR 97402-0414, USA
(NH2-Phe)2-R110 = aminopeptidase metalloproteinase substrate (MW: 624.62), laboratory synthesis
AO = acridine orange (MW: 301.83), Nr.4539, Polysciences, Warring ton, PA, USA
PI = propidium iodide (MW: 668.4), Nr.P-4170, Sigma, St.Louis, Miss., USA, Germany: Sigma Chemie GmbH, D-82041 Deisenhofen
fluoresceine = (MW: 376.3), Serva, Nr.21572 D-69115 Heidelberg, Germany

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6.2 REAGENTS:

PMA = phorbol-myristate-acetate (MW: 662.0), Nr.P8139, Sigma, St.Louis, Miss., USA, Germany: Sigma Chemie GmbH, D-82041 Deisenhofen
H2O2 = perhydrol 30% (MW: 34.01), Nr.7210, Merck, D-27476 Darmstadt, Germany
ionomycin = calcium ionophore (MW: 709.0), Nr.407950, Calbiochem, D-65812 Bad Soden, Germany
A23187BR = calcium ionophore (MW: 602), Nr.100107 Calbiochem, D-65812 Bad Soden, Germany
gramicidin D = H+-ionophore (MW: ...), Nr.G-5002, Sigma, St.Louis, Miss., USA, Germany: Sigma Chemie GmbH, D-82041 Deisenhofen
Z-Phe-Ala-CHN2 = cathepsine B,H,L, cysteine proteinase inhibitor (diazo-methyl-ketone, MW 394.4), Nr.N-1040, Bachem, D-69115 Heidelberg
Z-Leu-CHN2 = aminopeptidase, metalloproteinase inhibitor (chloro- methyl-ketone, MW 200.11), Nr.1260, Bachem, D-69115 Heidelberg
DFP = di-isopropylfluorophosphate, cathepsin G serine proteinase inhibitor, (MW 181,15), Nr.D12,600-4 Aldrich-Chemie, D-89555 Steinheim
EDTA = 0.2 M pH 7.2 (di-Na EDTA, (MW: 372.25), Nr.8418 Merck, D- 64271 Darmstadt, Germany
CaCl2 = 0.3 M CaCl2.2H2O solution aqua dest. (MW: 110.99), Nr.2382, Merck, D-64271 Darmstadt, Germany
HgCl2 = mercury-II-chloride (MW: 200.59), Nr.4404 Merck, D-64271 Darm- stadt, Germany
particles = PolybeadTM Carboxylate Microspheres(2.5% Latex-Solids) , UV excitable (365nm) Nr.18340 4.29um BB, 488nm FITC-excitable Nr.16592 4.30um, YG Polysciences Ltd., Warrington, PA, USA, Germany: Handelsstr.3, Postfach 1130, D-69208 Eppelheim, Tel: 06221/765767, Fax: 06221/764620
NaCl = Natriumchlorid (MW: 58.44), Nr.6404 Merck, D-64271 Darmstadt, Germany HEPES = N-(2-Hydroxyethyl)-piperazine-N'-2-ethane-sulfonic acid (MW: 238.3), Nr.25245 Serva, D-69115 Heidelberg, Germany
HBS-buffer = 0.15M NaCl2, 5mM HEPES pH 7.35
DMF = di-methylformamide, (MW: 73.10) Nr.7156 J.T.Baker Chemicals, Deventer, Holland
DMSO = di-methylsulfoxide, (MW: 78.13) Nr.D-8779 Sigma Chemicals, St.Louis, Missouri, USA

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6.3 UV-EXCITATION (365nm) ASSAYS

HBO-100 lamp, HeCd or Ar-Laser

6.3.1 INTRACELLUAR pH/ESTERASE ACTIVITY:

 ADB: 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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6.3.2 INTRACELLULAR CALCIUM:

INDO1/AM: 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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6.3.3 FREE GLUTATHIONE/FREE PROTEIN SH-GROUPS:

OPT: 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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6.3.4 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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6.3.5 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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6.4 BLUE LIGHT FLUORESCENCE EXCITATION

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

6.4.1 CELLULAR ENERGY:

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

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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6.4.2 CELLULAR ENERGY:

R123: mitochondrial membrane potential rhodamine123 MW: 380.8 D

assay:
250ul cell suspension
+5ul R123 .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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6.4.3 METABOLIC STATE:

SNARF1/AM: intracellular pH, SNARF1-acetoxymethylester MW: 568 D
- predilute: .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 .11ug/ml (.2uM), PI 40ug/ml (60uM))

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6.4.4 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 .45ug/ml (.4uM), PI 40ug/ml (60uM))

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6.4.5 OXIDATIVE BURST AND PEROXIDASE ACTIVITY:

DHR: sensitive H2O2 and peroxidase indicator system (8 x DCFH-DA)
dihydrorhodamine123 MW: 346.0 D
- DHR stock solution: 1.5mg/ml (4.0mM) in DMF, freeze at: -180oC
- predilute: 5ul DHR 1.5mg/ml in DMF + 500ul 2mg/ml PI in HBS (DHR 1/100, 15mg/ml,40mM, PI 2mg/ml 3mM, DMF 1%), freeze at: -180oC
- PMA stock solution: .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 .5%)
- H2O2: 1/200 (50mM) prediluted from Perhydrol (Merck) with HBS

assay1: spontaneous oxidation
250ul cells
+5ul DHR 15ug/ml, PI 2mg/ml, DMF 1%
- incubate 15min at 22oC
- measure (DHR 0.3ug/ml (.86uM), PI 40ug/ml (60uM), DMF .02%)

assay2: PMA stimulation (positive control)
250ul cells
+5ul DHR 15ug/ml, PI 2mg/ml, DMF 1%
- incubate 15min at 22oC or 37oC
+5ul PMA 5uM, DMF .5% in HBS
- incubate 10min at 22oC or 37oC
- measure (DHR 0.3ug/ml (.86uM), PI 40ug/ml (60uM), DMF .03%)

assay3: peroxidase activity
250ul cells
+5ul DHR 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 (DHR 0.3ug/ml (.86uM), H2O2 (1mM), PI 40ug/ml (60uM), DMF .02%)

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6.4.6 OXIDATIVE BURST AND PEROXIDASE ACTIVITY:

DCFH-DA: H2O2 formation and peroxidase activity (less sensitive)
dichlorofluorescin-diacetate MW: 487.3 D
- DCFH-DA stock solution 4.87mg/ml (10mM) in DMF
- predilute 10ul DCFH-DA stock solution with 90ml with 2mg/ml PI in HBS daily (DCFH-DA 1/10, 1mM, PI 2mg/ml 3mM, DMF 10%)
- PMA stock solution: .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: .5%)
- H2O2: 1/200 (50mM) prediluted from Perhydrol (Merck) with HBS

assay1: spontaneous oxidation
250ul cell suspension
+5ul DCFH-DA .487ug/ml, PI 2mg/ml in HBS, DMF: 10%
- incubate 15min at 22oC
- measure (DCFH-DA 9.74ug/ml (20uM), PI 40ug/ml (60uM), DMF: .2%)

assay2: PMA stimulation (positive control)
250ul cell suspension
+5ul DCFH-DA 1/10 .487mg/ml, PI 2mg/ml in HBS, DMF: 10%
- incubate 15min at 22oC or 37oC
+5ul PMA 5uM, DMF .5% in HBS
- incubate 10min at 22oC or 37oC
- measure (DCFH-DA 9.74ug/ml (20uM), PMA 100nM, PI 40ug/ml (60uM), DMF .2%)

assay3: peroxidase activity
250ul cells
+5ul DCFH-DA 1/10 .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 .2%)

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6.4.7 OXIDATIVE BURST:

HE: O2- radical formation/general intracellular oxidation, hydroethidine MW: 315.0 D
- 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 .07%)

assay2: PMA stimulation (positive control)

assay3: peroxidase activity
(both assays similarly as above)

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6.4.8 OXIDATIVE BURST:

SIMULTANEOUS O2- and H2O2 formation + intracellular oxidation DCFH-DA+HE - 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 487ug/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 .27%)

assay2: PMA stimulation (positive control)

assay3: peroxidase activity
(both assays similarly as above)

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6.4.9 PROTEASES:

6.4.9.1 R110 Substrate Solutions:

R110: protease substrates
Stock solutions (4mM):
- (Z-Arg2)2-R110, 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, 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, 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, aminopeptidase: 2.22 mg/ml in DMF, dilute 1/20 with 2mg/ml PI in DMF to .111 mg/ml (0.2mM)
- (NH2-Phe)2-R110, aminopeptidase: 2.49 mg/ml in DMF, dilute 1/20 with 2mg/ml PI in DMF to .125 mg/ml (.2mM)

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6.4.9.2 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).

6.4.9.3 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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6.4.9.4 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 .2mM in HBS
- incubate 15min at 20oC or 37oC
+5ul substrate .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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6.4.9.5 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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6.4.10 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 .83ug/ml (2uM), PI 40ug/ml (60uM))

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6.4.11 DNA/RNA:

AO: vital DNA/RNA acridine orange MW: 301.83 D

assay:
250ul cell suspension
+5ul AO .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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7. LITERATURE REFERENCES

7.1 Flow Cytometric Methods:

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

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7.2 Cytostatic Drug Assays:

1. Valet G., Warnecke H.H., Kahle H.: Cytostatic drug testing by flow-cytometry. Paesel, Frankfurt, 1984
2. Valet G., H.H.Warnecke, H.Kahle: New possibilities of cytostatic drug testing on patient tumor cells by flow cytometry. Blut 49,37-43, (1984)
3. Neubauer A., R.Sauer, G. Valet: Cytostatic drug testing in human leukemias by multiparameter flow-cytometry. Blut 55,433-445,(1987)
4. Neubauer A., G.Valet, D.Huhn: Flow-cytometric determination of intracellular pH, esterase activity and cell volume in human leukemic cell lines following in-vitro incubation with cytostatic drugs. J.Anal.Cell.Pathology 2,49-58,(1989)
5. Hasman M., G.Valet, H.Tapiero, K.Trevorrow, T.Lampidis: Membrane potential differences between adriamycin sensitive and resistant cells as measured by flow cytometry Biochem.Pharmacol. 38,305-312,(1989)
6. Lampidis Th.J., C.Castello, A.Giglio, B.C.Pressman, P.Viallet, K.W.Trevorrow, G.K.Valet, H.Tapiero, N.Savaraj: Relevance of the chemical charge of rhodamine dyes to multiple drug resistance. Biochem.Pharmacol. 38,4267-4271,(1989)
7. Lampidis Th.J., N.Savaraj, G.K.Valet, K.Tevorrow, A.Fourcade, H.Tapiero: 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)
8. Wulf G., H.Falk, U.Weiershausen, G.Valet: 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)

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7.3 Data Analysis:

1. Valet G., L.Rüssmann, R.Wirsching: 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)
2. Valet G., M.Valet, D.Tschöpe, H.Gabriel, G.Rothe, W.Kellermann, H.Kahle: 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)

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7.4 Clinical Applications:

1. Rothe G., W.Kellermann, G.Valet: 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
2. Rothe G., W.Kellermann, G.Valet: 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)
3. Liewald F., N.Demmel, R.Wirsching, H.Kahle, G.Valet: Intracellular pH, esterase activity and DNA measurements of human lung carcinomas by flow-cytometry Cytometry 11,341-348,(1990)
4. Liewald F., L.Sunder-Plassmann, H.Dienemann, H.Kahle, G.Wulf, G.Valet: 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)
5. Liewald F., R.Hatz, M.Storck, K.H.Orend, M.Weiss, G.Wulf, G.Valet, L.Sunder-Plasmann: Prognostic value of deoxyribonucleic acid aneuploidy in primary non small-cell lung carcinomas and their metastases. J.Thoracic Cardiovasc.Surgery 105,1476-1482,(1992)
6. Rothe G., W.Kellermann, J.Briegel, B.Schaerer, G.Valet: 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

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7.5 Marine Plankton:

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

For problems or comments, please contact:
G.Valet E-mail: valet@vms.biochem.mpg.de, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany, Tel: +49/89/8578-2518, -2525, Fax: +49/89/8578-2563
INTERNET address: http://www.biochem.mpg.de/valet/cellbio.html, Last update: Feb.26, 1996