Detection and purification of rare Ca2+responders by
fixed-time flow cytometry
Attila Tárnok and Henning Ulrich
List of Contents
1. Introduction
7. Results
2. Materials
8. Troubleshooting
3. Procedure
9. References
4. Calcium Measurement
10. Suppliers
5. Fixed Time Device
11. Figures
6. On Line Measurements
___________________________________________________________________________
1. Introduction
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Flow cytometrical analysis of cells stained with the calcium-sensitive dye indo-1 (Haugland,
1992) is now widely used for the measurement of cytosolic calcium [Ca2+]i of cells in response
to external signals (Tárnok, 1996; 1997; Ulrich et al., 1996). These changes occur typically
after stimulation of membrane bound receptors leading to an opening of intracellular calcium
stores (Jin et al., 1994) and/or to a change of the activity of calcium channels located in the
plasma membrane (Ulrich et al., 1996). These changes in [Ca2+]i are often transient, lasting in
the second to minute time range.
Measurement of [Ca2+]i can be performed in two different ways: By on-line analysis (Omann
et al., 1985; Tárnok, 1996; Ulrich et al., 1996) and by fixed-time analysis (also called timewindow analysis) (Dunne, 1991; Tárnok, 1996; 1997). For the detection of fast responses, the
cells are stimulated by the injection of the ligand solution. A rapid subsequent increase of
sample pressure ensures a rapid passage of the stimulated cells to the laser intercept. The lag
period can be adjusted normally to be less than 1 s. The disadvantage of this method is that the
rapid increase in pressure during the ligand injection may activate pressure-sensitive ion
channels that overlay the specific ligand-induced response. Ligand-specific changes in [Ca2+]i
of cells expressing pressure-sensitive ion channels can be analyzed easily by fixed-time
measurements (Tárnok, 1996). Fixed-time analysis is also a sensitive method to analyze
heterogeneous cell populations where only a subpopulation responds to ligand application
(Tárnok, 1997).
We describe here the protocol for fixed-time flow cytometry that was used to quantify small
changes in [Ca2+]i in a heterogeneous population of pressure-sensitive cells. As examples for
the quantification of small responses, the neuropeptide head activator (Schaller and
Bodenmüller, 1981) -induced increase in [Ca2+]i of neuroblastoma X glioma NH15-CA2 cells
(Heumann et al., 1979) and P19 cells that had been stimulated to neuronal differentiation with
retinoic acid were used (Niemann and Schaller, 1996). HA was shown to lead to an increase
of [Ca2+]i in a subpopulation of 10–15 % of NH15-CA2 cells. HA also stimulated an increase
of [Ca2+]i in P19 cells that had been induced to neuronal differentiation by treatment with
retinoic acid. Less than 10 % of the P19 cell population responded to HA with an increase of
[Ca2+]i.
The results presented show that ligand-induced alterations in only a small subpopulation of
NH15-CA2 cells can be quantified exactly by fixed-time analysis since the ligand-specific
response is not overlaid by the pressure-induced activation of ion channels. The method is also
suitable for quantifying the HA-induced response in retinoic acid-induced P19 cells.
Using fluorescence-activated cell sorting in combination with fixed-time flow cytometry,
responders from heterogeneous cell cultures can be sorted aseptically and cultivated for further
investigations (Julius et al., 1988; Schieren and MacDermott, 1988; Tárnok, 1997).
2. Materials

Cells
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 Subconfluent cultures from embryonic and differentiated P19 cells (kindly provided by Dr S
Niemann, Center for Molecular Neurobiology, University Hospital of Hamburg, Germany)
and NH15-CA2 cells.
 NIH/3T3 murine fibroblasts cotransfected with total genomic DNA of the rat pituitary gland
cell line GH3 and neomycin resistance gene (cells provided by Dr W Meyerhof, Institute
for Cell Biochemistry and Clinical Neurobiology, Hamburg, Germany; Richter et al.,
1991). Transfectants contain a high fraction of up to 1 % donor derived DNA in order to
increase the probability of cells expressing intact neuropeptide and -transmitter receptors.
 Stimulate with neuropeptides and -transmitters that are known to bind G-protein coupled
receptors and induce an increase of [Ca2+]i: bradykinin, bombesin, arginine vasopressin,
oxytocin, neurotensin, substance-P, noradrenalin and/or 5-hydroxytryptamine (5-HT or
serotonin; all compounds from Sigma Chemical, St.Louis, MI) (Berridge, 1993; Tárnok,
1997) and head-activator (HA; Bachem, Bubendorf, Switzerland) (Ulrich et al., 1996).
Store as 1 mM stock solutions in distilled water at -20°C.
 Cell cultivation at 37 °C, 5 % CO2.
Cell culture media
 NH15-CA2: DMEM medium supplemented with 10 % fetal calf serum, 2 mM glutamate and
100 IU/ml penicillin/streptomycin (all from Gibco), and buffered with 10 mM HEPES, pH
7.2 (Sigma).
 P19: use instead of DMEM an equal mixture of DMEM and Nut mix F-12 (Gibco).
 NH15-CA2, P19: Defined medium (Bottenstein and Sato, 1979): DMEM or DMEM and Nut
mix F-12 (1:1) supplemented with 5 µg/ml insulin, 30 µg/ml transferrin, 20 µM
ethanolamine, 30 nM sodium selenite, 100 IU/ml penicillin/streptomycin, 2 µM sodium
pyruvate and 1 % non-essential amino acids.
 NIH/3T3: RPMI 1640 (Sigma Chemical) supplemented with 10 % fetal calf serum, 2 mM
glutamate and 100 IU/ml penicillin/streptomycin and buffered with 10 mM HEPES, pH 7.2.
Transfectants are cultivated in medium with the neomycin analogue G418 (400 µg/ml,
Gibco; Richter et al., 1991).
Chemicals
 Indo-1 AM (Molecular Probes, Eugene, OR) dissolved in acetone at 1mM (1mg/ml),
vacuum dried and frozen in 20 µg aliquots and stored at -20°C. For staining indo-1 is
diluted to a final concentration of 8 µM (5 µM for NIH/3T3) with 1 % DMSO and 0.2 %
pluronic F-127 (Molecular Probes).
 Propidium iodide (PI, Molecular Probes) for dead cell discrimination is stored at 1mg/ml in
distilled water at 4 °C and used at a final concentration of 1-5 µg/ml.
 Calcium buffer CALBUF-2 (World Precision Instruments, Sarasota, FL).
 Calibration cocktail: HEPES, sodium azide, deoxy-D-glucose, carbonyl-cyanide mchlorophenyl-hydrazone (CCCP), nigericin, ionomycin, Br-A23187 (all reagents from
Sigma).
Flow cytometer
 TygonTM tubes as sample lines (0.25 mm I.D., Reichelt Chemietechnik, Heidelberg,
Germany), T-junction (Cytek Dev., Fremont, CA), tubing and connectors for the sample
pressure and a waterbath are required, Nylon gauze (mesh size 50 µm, Bückmann,
Mönchengladbach, Germany).
 On-line injection system: e.g. time-zero module (Cytek Dev., Fremont, CA; Omann et al.
1985).
 Optical filters: dichroic filter (TY312, Schott, Mainz, Germany), bandpass filters: 400 nm
(NAL 400, Schott) and 525 nm (Coulter Corp.). Other supplier: Omega Optical, UK.
 Software: MDADS data analysis system (Coulter Corp.), data analysis system DAS
(Beisker, 1994), SigmaPlot (SPSS, Arlington, VA)
3. Procedure
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Cell lines
NH15-CA2 and P19 cells
1. Culture NH15-CA2 and P19 cells in their respective medium. Stimulate embryonic P19
cells to neuronal differentiation by plating into non-adhesive tissue culture flasks with
defined medium to induce spontaneous aggregation. Retinoic acid (Sigma Chemicals) at a
final concentration of 1 µM is added 24 h and 48 h after plating. Transfer the cells after 3
days into adhesive culture flasks containing DMEM medium with 10 % FCS. After 6 days
of culture retinoic-acid stimulated P19 cells express neuronal markers and can be
stimulated by the neuropeptide head activator.
2. Keep NH15-CA2 cells, embryonic and differentiated P19 cells before the experiments
overnight in defined medium.
NIH/3T3 transfectants
1 Transfer transfected cells prior to sorting into G418-free RPMI medium with low (1-2 %)
fetal calf serum. Reduction of serum level reduces background of spontaneously firing
cells (Tárnok, 1997).
2. Stimulate cells either with single substances or with a cocktail. The final concentration of
each neuropeptide and neurotransmitter in our experiments was 1 µM. In the sorting
experiments bradykinin and 5-HT were omitted from the ligand cocktails as these receptors
are also present on the non-transfected cell line (Tárnok, 1997).
4. Calcium measurements
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Staining with indo-1 AM
1. Cells are harvested by scraping, collected by centrifugation (300 x g, 5 min) and
resuspended in defined medium at a final density of 106 cells/ml.
2. Stain with 8 µM indo-1 (5 µM indo-1 for NIH/3T3) in its cell-permeant form as
acetoxymethyl ester (indo-1 AM, Molecular Probes) in the presence of 1 % DMSO and 0.2
% of the non-ionic surfactant pluronic F-127 in defined medium at a concentration of 106
cells/ml.
3. Wash the cells twice with defined medium or RPMI, dilute to a density of 105 cells/ml and
keep them in the dark at room temperature until analysis.
4. Filter cell suspension prior to analysis through a gauze of 50 m mesh size to remove
clumps and warm the suspension up to 37oC for 5 min. Perform all further steps at 37oC.
Calibration of indo-1 fluorescence
Calibration curves are constructed using Ca2+ buffer solutions in the range from 10-8 to 10-4 M
Ca2+ (CALBUF-2, World Precision Instruments; Szöllösi et al., 1991).
1. Indo-1 loaded cells are centrifuged and resuspended at a density of 105 cells/ml in calciumfree buffer containing 10 mM HEPES, pH 7.2, 5 mM sodium azide, 5 mM deoxy-Dglucose, 20 µM CCCP, 5 µg/ml nigericin, 4 µionomycin, and 5 µ Br-A23187.
2. Add pretreated cells to different calcium buffer solutions and incubate for 30 min at 37 °C
before measurement. Cells should be measured within 1 h after incubation.
Instrumentation
Changes in the cytosolic free calcium concentration [Ca2+]i are detected ratiometrically with
the calcium-sensitive dye indo-1.
1. For excitation of indo-1, the Argon laser is tuned to 300 mW power output at all-line UV
emission mode.
2. The emitting indo-1 fluorescence is split by a dichroic filter and collected at 400 and 525
nm (Rabinowitch and June, 1994).
3. The parameters measured are forward angle light scatter, the fluorescence emission
intensities of indo-1, the ratio of the both indo-1 fluorescence intensities, and time.
4. The analogue signals are digitized on an 8-bit ADC board, and the 400/525 nm
fluorescence ratio is calculated on-line. The data are transferred to an IBM-compatible
computer using the Gateway Software (Coulter Corp.). Analysis of the acquired list mode
data is done with the DAS software package (Beisker, 1994).
Measurement of indo-1, gating and data analysis
1. Data acquisition is triggered by forward angle light scatter signal (Fig.1.A).
2. Cells are gated on forward angle light scatter and logarithmic 400nm fluorescence intensity
to measure only viable stained cells (Gate 1, Fig.1.A).
3. Cells with low or off-scale linear fluorescence intensity are discarded (Gate 2, Fig.1.B).
Eventually dead cells that are stained with propidium iodide for 5 min at room temperature
can be discarded (Gate 3, Fig. 1.C).
4. From the residual gated indo-1 ratio the intracellular free calcium concentration [Ca2+]i can
be calculated.
5. Percentage of responders is calculated as percentage of cells more than two standard
deviations above the mean ratio of unstimulated cells.
5. Fixed-time device
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1. Connect tubing of the fixed-time device as schematically depicted in Figure 2. Connect
device to the cell sorter (EPICS751, Coulter Corp.) or to any other commercially available
flow cytometer.
2. For fixed-time measurements cell suspension and ligand solution are kept in separate vials.
The cell suspension and ligand solution are driven by the sample pressure through sample
lines into a T-junction. In the T-junction the cells mix with the ligand solution and flow
through the connecting tube to the laser intercept.
3. The two pinch valves A and B at the sample lines are used to equalize the sample-flow rate
from both vials.
4. After connecting the fixed-time device to the flow cytometer, run distilled water followed
by medium before running the cells.
Calibration of the incubation time
The incubation time of the cells with the ligand solution depends on the sample-flow rate and
the systemic or line pressure. Sample-flow rate is proportional to the differential sample
pressure and the length of the connecting tube and increases with rising line pressure (Fig.
3.A).
1. Measure at different sample pressures and constant line pressure the sample flow-rate, this
yields a linear relationship of flow rate and sample pressure (Fig. 3.A).
2. Calculate at a certain sample pressure the incubation time with the calculated volume after
the T-junction (i.e. volume of connecting tube plus volume between adapter at the flow
cytometer and laser intercept). This yields a linear relationship with the sample pressure
(Fig. 3.B).
3. Verify the calculated incubation time with a timer after opening pinch valve C (Fig. 1).
Normally there is a good correlation between calculated and measured incubation time
(Fig. 3.C).
In our system the minimum (calculated) incubation time was 0.74 s at 0 cm connecting tube
length. On bench top flow cytometers the sheath pressure is fixed and sample pressure can
only be altered stepwise (e.g. FacsCalibur, Becton & Dickinson). In these machines only
discrete incubation times can be achieved.
Aseptic sorting
1. Clean sample and sheath fluid lines with detergent (Coulter Cleanser, Coulter Corp.) and
rinse with sterile distilled water for 20 min each. Use sterile isotonic solution (e.g. 0.9 %
NaCl) as sheath fluid. Sodium azide, formaldehyde or detergents must be omitted.
2. Collect sorted cells in tubes containing medium, 10 % FCS, G418 (400 µg/ml), and 200 IU
antibiotics. Wash twice with medium and seed cells into culture flasks with G418
containing medium.
3. Cultivate cells and reanalyze after 2-3 weeks of cultivation.
When low numbers of responders are expected the non-viable (i.e. membrane damaged)
cells are stained additionally with PI (see Fig. 1 B).
6. On-line Measurements
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On-line stimulation can be measured on the time-zero module (Cytek Dev., Fremont, CA).
1. Measure baseline indo-1 ratio of unstimulated cells for a period of 30 – 60 s using a
constant low sample pressure provided e.g. by a purified nitrogen source.
2. Stimulate cells by injection of 100 µl of ligand solution into the cell suspension in a total
volume of 1 ml (control experiments: injection of 100 µl defined medium). The time delay
between stimulation and the appearance of the first stimulated cells should be minimized to
be less than 2 s by increasing the sample pressure during the ligand injection.
3. Monitor the kinetics for the duration of the calcium transient.
7. Results
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Pressure-sensitive NH15-CA2 cells
Figure 4 A shows a typical on-line stimulation of NH15-CA2 cells by injection of medium or
HA. Injection of medium or HA results in a transient increase of [Ca2+]i. The specific increase
of [Ca2+]i. that is induced by HA application (Ulrich et al., 1996) is almost overlaid by the
unspecific response due to the activation of pressure-sensitive ion channels.
Stimulation of NH15-CA2 cells using the fixed-time device at a constant sample pressure
results in a stable [Ca2+]i. at a certain setup. Defined medium as ligand solution does not
induce any changes in [Ca2+]i.. The analysis of the HA-induced changes reveals an increase of
cells showing an increased [Ca2+]i. level from 7.8 % to 17.6 % after 8.7 s incubation between
cell suspension and HA (Fig. 4.B). The elevated [Ca2+]i. of HA-stimulated NH15-CA2 cells is
transient returning to basal levels after 60 s (data not shown).
In Figure 5 the residual calcium kinetics measured on-line (Fig. 5.A) and by fixed-time
flow cytometry (Fig. 5.B) with defined medium and HA are shown. This comparison shows
that injection of defined medium leads to an elevation of [Ca2+]i. with a 100 nM peak value.
This response is due to the applied pressure during ligand application and not to compounds in
the defined medium used for stimulation; the mixing of the cell suspension with defined
medium did not lead to any changes of [Ca2+]i. when analyzed by the fixed-time method (Fig.
5.B). The specific effect of HA that could hardly be quantified by on-line measurements was
estimated to be 50 nM above baseline.
Differentiation of P19
P19 cells that had been stimulated by treatment with retinoic acid to neuronal differentiation
express the HA receptor and respond to HA application with a transient increase of [Ca2+]i
(Niemann and Schaller, 1996). Figure 6 compares the stimulation of embryonic and neuronal
differentiated P19 cells with defined medium and HA using on-line stimulation (Fig. 6.A) and
the fixed-time device (Fig. 6.B). Stimulation of embryonic P19 cells using either defined
medium or HA does not result in any change of [Ca2+]i. The results obtained by on-line and
fixed-time analysis were identical (Fig. 6, upper lane). On-line analysis of neuronal
differentiated P19 cells that are stimulated by injection of defined medium reveal a rise of
cells showing elevated [Ca2+]i levels that was similar to the [Ca2+]i of cells that had been
stimulated by injection of HA (Fig. 6, lower lane). The pressure-induced increase of [Ca2+]i
overlays the specific response due to HA application. Using the fixed-time device, the HAinduced rise of cells showing elevated [Ca2+]i levels increased from 7.6 to 12.8 %.
Sorting of transfectants
Unsorted transfected NIH/3T3 cells do not show detectable response to a single ligand or to
ligand cocktails (Fig. 7.A). The background fraction was initially between 1 % and 5 % but
was around 0.5 % if dead and membrane damaged cells were excluded by propidium iodide
staining (Tárnok, 1997). Fixed-time sorting was done with a neurotransmitter cocktail. 10,000
Cells were sorted and subsequently cultivated for two weeks. During sorting the sample
pressure was varied so that cells responding between 5-20 s after stimulation were collected.
The first sorted culture contained 4 - 5 % responders (Fig. 7). In the second sorting ~500 cells
were sorted and cloned. 25 of the residual clones were reanalyzed after 3 weeks of cultivation.
Six of these clones responded to the stimulatory cocktail (Fig. 7.A).
Figure 7.B shows the dose response curve of clone No.116 to oxytocin application analyzed
by fixed-time flow cytometry in the range of 10-20 s incubation time. The clone was nonresponsive to other compounds of the cocktail (not shown) except bradykinin (Fig. 7.C). For
clone 116 optimal concentrations were around 20 mM for oxytocin and 10 µM for bradykinin.
The initial frequency of oxytocin responders can be estimated from the percentage of vital
cells with increased [Ca2+]i in the unsorted culture, the percentage of responders after the first
sort and the frequency of sensitive cells after cloning (2 of 25) (Tárnok, 1997). In this example
it was estimated that the initial frequency of oxytocin sensitive cells was about 1:50,000 to
3:1,000,000 (Tárnok, 1997).
8. Troublesshooting
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 Cell storage
Prior to experiments cells should be stored at room temperature in the dark. Storage on ice and
rewarming immediately (i.e. 10-20 min) prior to the experiment might lead to nonresponsiveness or massive spontaneous calcium bursts.
 Staining
Some cell lines do not stain brightly for indo-1. Staining can be increased by increasing indo-1
and/or F-127 concentrations but it should be taken into account that high indo-1
concentrations might buffer [Ca2+]i and poison the cells. DMSO concentrations higher than
1% can stimulate cells and modify their membrane integrity. In cells where intracellular
esterase activity is low or absent the cleavage of the acethoxymethylester from indo-1 is
impeded. These cells are better stained with indo-1 and a brief hypotonic treatment induces the
uptake of the dye into the cells.
 Long fixed-time incubation time
Analysis of incubation times greater than 3 min makes a connecting tube length of more than
70 cm necessary. These measurements are inconvenient, lead to mechanical stress, and are
better performed by on-line analysis.
 Elevated background levels
The flow cytometer should be well maintained and cleaned as, in particular, dried saline
deposits around the nozzle assembly could deflect the particle jet and lead to inaccurate results
and an increase in background noise.
Other reasons leading to inaccurate measurements are too high counting rates at low sample
pressure and cells that are forming clumps during the measurements. Therefore the cells
should be filtered through gauze and according to the chosen sample pressure appropriately
diluted immediately before the measurements. Optimal count rates are in the range of 1001,000 events/s. If the same cell suspension is used for measurements that take several hours,
the cells should be kept at room temperature and in the dark.
 Lack of stimulation
Failure of stimulation induced by the respective ligand may result from tubing that has been
contaminated by the ligand solution during former measurements. For „sticky“ stimuli the
sample lines should be washed with organic solvent solution (e.g. ethanol or methanol) and
sample lines and connecting tube has to be replaced on regular terms.
Failure of stimulation may also result from a not correctly chosen time window. The kinetics
of the stimulation reactions should firstly be monitored by on-line measurements to determine
the time point of maximal stimulation.
If questions arise concerning special applications of the above described protocols beside of
the authors the following cytometry mailing lists may be contacted by e-mail:
cytometry@flowcyt.cyto.purdue.edu (ISAC, International Society for Analytical Cytology)
zytometrie@medizin.uni-leipzig.de (DGfZ, Deutsche Gesellschaft für Zytometrie).
Useful tips may be found in the Cytorelay Node of the Max-Planck-Institute for Biochemistry,
Martinsried, Germany (http://www.biochem.mpg.de/groups/valet/reagent1.html)
We wish to thank Dr. S Niemann for providing undifferentiated and neuronal differentiated P19
cells, Dr. W. Meyerhof for providing untransfected and transfected NIH/3T3 fibroblasts.
We also want to thank Prof.Dr. H. C. Schaller, Center for Molecular Neurobiology, University
Hospital of Hamburg, Germany, for support.
9. References
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
Beisker W (1994) A new combined integral-light and slit-scan data analysis system
(DAS) for flow cytometry. Comput.Methods. Programs Biomed 42:15-26. return

Berridge MJ(1993) Inositol triphosphate and calcium signaling. Nature 361:315-325.
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
Bottenstein JE, Sato G (1979) Growth of a rat neuroblastoma cell line in serum-free
supplemented medium. Proc Natl Acad Sci USA 76: 514-517. return

Dunne JF (1991) Time window analysis and sorting. Cytometry 12: 597-601. return

Haugland R P (1992) Handbook of fluorescent probes and research chemicals. Molecular
Probes Inc., Eugene, OR, USA.. return

Heumann R, Öcalan M, Kachel V, Hamprecht B (1979) Clonal hybrid cell lines
expressing cholinergic and adrenergic properties. Proc Natl Acad Sci USA 76: 4674 –
4677. return

Jin W, Loh NM, Loh HH, Thayer SA (1994) Opioids mobilize calcium from inositol
1,4,5-triphosphate-sensitive stores in NG108-15 cells. J Neurosci 14: 1920-1929. return

Julius D, McDermott A B, Axel R, Jessel TM (1988) Molecular characterization of a
functional cDNA encoding serotonin receptor. Science 241: 558-564. return

Niemann S, Schaller HC (1996) Head-activator and the neuroectodermal differentiation of
P19 mouse embryonal carcinoma cells. Neurosci Lett 207: 49-52. return

Omann GM, Coppersmith W, Finney DA, Sklar LA (1985) A convenient on-line device
for reagent addition, sample mixing, and temperature control of cell suspensions in flowcytometry. Cytometry 6: 69-73. return

Rabinowitch PS, June CH (1994) Intracellular ionized calcium, membrane potential and
pH. In: Ormerod MG (ed) Flow cytometry. A Practical Approach, IRL Press, Oxford,
pp.161-187. return

Richter D, Meyerhof W, Buck F, Morley SD (1991) Molecular biology of receptors of
neuropeptide hormones. In: Seifert G (ed.) Current topics in pathology: cell receptors.
Springer, Berlin, 1991, pp 117-139. return

Schaller HC, Bodenmüller H (1981) Isolation and amino acid sequence of a
morphogenetic peptide in hydra. Proc Natl Acad Sci USA 78: 7000-7004. return

Schieren I, MacDermott (1988) A flow cytometric identification and purification of cells
by ligand-induced changes in intracellular calcium. J Neurosci Methods 26: 35-44. return

Szöllösi J, Feuerstein BG, Hyun WC, Das, MK, Marton LJ (1991) Attachment of A127
human glioblastoma cells affects calcium signalling: a comparison of image cytometry,
flow cytometry, and spectrofluorometry. Cytometry 12: 707-716. return

Tárnok A (1996) Improved kinetic analysis of cytosolic free calcium in pressure-sensitive
neuronal cells by fixed-time flow cytometry. Cytometry 23, pp. 82-89. return

Tárnok A (1997) Rare event sorting based on changes in intracellular free calcium by fixed
time flow-cytometry. Cytometry 27: 65-70. return

Ulrich H, Tárnok A, Schaller HC (1996) Head-activator induced mitosis of NH15-CA2
cells requires calcium influx and hyperpolarization. J Physiol (Paris) 90: 85-94. return
10. Suppliers
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Suppliers
Product
American Type Culture Selection (ATCC)
P19 rat embryonal carcinoma
cells
12301 Parklawn Drive
Rockville, MS 20582-1776, U.S.A.
http://www.atcc.org
Bachem, Feinchemikalien AG
Hauptstr. 144, Bubendorf CH-4416,
Switzerland,
Phone and Fax: +41-61-9312333
Bückmann GmbH, Technische Gewebe
Head activator (customer
desired synthesis)
Nylon gauze of 50 M mesh size
Konstantinstr. 46
D-41238 Mönchengladbach
Phone: +49-2166-80023
Fax: +49-2166-82788
Cytec Development,
T-tube, time-zero module
46560 Fremont Boulevard, Suite 116,
Fremont, CA 94538, U.S.A.
Phone: +1-510657-0102
Fax: +1-510657-0151
Gibco Life Technologies
Box 6009
Gaithersburg, MD 20884-9980, U.S.A.
Phone: +1-301-840-8000
Fax +1-301-258-8238
http://www.lifetech.com
Molecular Probes,
PO Box 22010, Eugene, OR 97402-0469, U.S.A.
Phone: +1-541-465-8300
Fax: +1-541-344-6504
E-mail: tech@probes.com
Omega Optical
PO Box 573, 3 Grove Street
Brattleboro
Vermont 05302 USA
Phone: +1-802-254-2690
Fax: +1-802-254-3937
German Distributor: INSTRUMENTS S.A. GmbH.
Bretonischer Ring 13
D-8011 Grasbrunn 1; Germany
Phone: +49-89-4602051; Fax: +49-89-463197
Distributor in UK: Glen Spectra Ltd.
DMEM medium, antibiotics,
glutamate, sodium pyruvate,
non-essential amino acids
Indo-1 AM, pluronic F-127,
propidium iodide
optical filters, dichroic mirrors
2-4 Wigton Gardens
Stanmore, Middlesex HA7 1BG; UK
Phone: +44-81-2049517; Fax: +44-81-2045189
Reichelt Chemietechnik GmbH & Co
Tygon tubes, I.D. 0.25 mm
Engelstr.18
D-69126 Heidelberg, Germany
Phone: +49-6221-3125
Fax: +49-6221-301227
Schott Glaswerke
Box 2480
D-55014 Mainz
Fax: +49-61 31-66 20 00
Phone: +49-61 31-66-0
http://www.schott.de
Sigma Chemical Company
dichroic mirror,
optical filters
Box 14508
St. Louis, Missouri 63178-9916, U.S.A.
Phone: +1-314-771-5750
Fax: +1-314-771-5757
insulin, transferrin, sodium
selenite, retinoic acid,
HEPES, Br-A 23187, sodium
azide, 2-deoxy-D-glucose,
ionomycin, nigericin,
SPSS Federal Systems (U.S.)
Sigma Plot
Courthouse Place
2000 North 14th, Suite 320
Arlington, VA 22201
Phone: +1-703-527-6777
Fax: +703-527-6866
E-mail: corinne@spss.com
World Precision Instruments
Calcium buffer solutions
International Trade Center
175 Sarasota Center Boulevard
Sarasota, FL 34240-9258
Phone: +1-941-371-1003
Fax: +1-941-377-5428
E-mail: sales@wpiinc.com
Prof. Dr. Bernd Hamprecht
NH15-CA2 cells
Physiologisches, Chemisches Institut der
Universität Tübingen
Hoppe-Seyler-Str. 4
72076 Tübingen, Germany
E-mail: BerndHamprecht@uni-tuebingen.de
Dr. Wolfgang Beisker
GSF-AG Durchflusszytometrie
Ingolstädter Landstr. 1
D-85764 Neuherberg Germany
E-mail: Beisker@gsf.de
DAS software package
Abbreviations:
HA:
Head-activator neuropeptide
[Ca2+]i:
intracellular free calcium concentration
PI
Propidium Iodide
FCS
fetal calf serum
CCCP
carbonyl-cyanide m-chlorophenyl-hydrazone
10. Figures
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Figure 1: Gating of indo 1 stained cells. Original dot-plots of NH15-CA2 cells before (low
[Ca2+]i) and after (high [Ca2+]i) stimulation with bradykinin (1 µM). By Gate 1 unstained
cells and debris are discarded, by Gate 2 cells with low or off-scale linear indo-1
fluorescence that would produce irrelevant ratio values are excluded. By Gate 3 dead, i.e.
propidium iodide (PI) positive, cells can be excluded.
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.
Figure 2: Scheme of the fixed-time flow cytometer
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Figure 3: Typical calibration of the incubation time for the fixed-time device connected
to the EPICS 751 cell sorter (Coulter Corp.).
(A) Dependence of the sample-flow rate on the line pressure of the flow cytometer. Data are
original measurements at various line and differential pressures.
(B) Calculated incubation time using the fixed time device at two different connecting tube
lengths. The total sample volume after the T-junction was 3.213 µl and 9.59 µl for 4 cm
and 17 cm, respectively; the coefficients of correlation were r > 0.90.
(C) Comparison of measured and calculated incubation time with correlation coefficients of >
0.95.
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Figure 4: HA-induced effect on intracellular calcium concentration in NH15-CA2 cells
analyzed by on-line and fixed-time analysis.
(A) On-line measurements: indo-1 stained NH15-CA2 cells are stimulated by injection of
defined medium and HA (1 nM final concentration; injection indicated by arrows). The
lag time between injection and appearance of stimulated cells is less than 2 s; the time
resolution is 1.5 s per channel. At each time point about 300 cells are measured.
(B) Fixed-time measurements: The defined medium (left row) and HA (1 nM)-induced
responses (right row) are determined. Indo-1 ratio distributions are shown for different
incubation times. The length of the connecting tube is 17 cm, and the differential sample
pressures are 2,7, and 15 psi for calculated incubation times of 8.7, 13.7, and 25 s,
respectively. The indo-1 fluorescence ratio is plotted vs. cell number. The lines in the
figures show the region in which cells are regarded as responders.
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Figure 5: Mean calcium kinetics of defined medium and HA on NH15-CA2 cells
measured on-line and fixed-time.
(A) NH15-CA2 cells are stimulated by defined medium (upper panel) or HA (1 nM, lower
panel) injection.
(B) Fixed-time measurements are performed at incubation times of 8.7, 13.7, and 25 s. The
differential sample pressures are 2, 7, and 15 psi. The length of the connecting tube is 17
cm. Data are acquired at a time resolution of 1.5 s. The calcium content of about 300 cells
is averaged per time point.
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Figure 6: HA-induced effect on intracellular calcium concentration in embryonic and
neuronal differentiated P19 cells analyzed on-line or fixed-time.
(A) On-line measurements: Embryonic (upper panel) and neuronal differentiated P19 cells
(lower panel) are stimulated by injection of defined medium and HA (1 nM final
concentration; injections indicated by arrows).
(B) Fixed-time measurements: Defined medium and HA-induced responses on embryonic
(upper panels) and differentiated P19 cells (lower panels) are shown. The differential
sample pressure is 5 psi and the connecting tube length is 17 cm (incubation time of 10 s).
The indo-1 fluorescence ratio is plotted vs. cell number. The lines in the figures show the
region in which cells were regarded as responders.
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Figure 7: Sorting and cloning of transfected NIH/3T3 mouse fibroblast following
neuropeptide and neurotransmitter induced calcium flux by fixed-time flow
cytometry.
(A) Percentage of responders to the neuropeptide and neurotransmitter cocktail used for
sorting is shown for unsorted, first sorted and second time sorted and cloned transfectants.
(B) Dose response of clone 116 to oxytocin
(C) Dose response to bradykinin.
The data shown in (B) an (C) are mean values of at least three experiments + SD.
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______________________________________________________________________
Correspondence to Dr. Attila Tarnok, Ph.D., Pediatric Cardiology, Herzzentrum Leipzig
GmbH, University Hospital www.herzzentrum-leipzig.de,
Russenstr.19, D-04289 Leipzig, Germany.
Phone: *49-341-865-2430, Fax: *49-341-865-1405,
e-mail tarnok@server3.medizin.uni-leipzig.de
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Dr. Henning Ulrich, Ph.D., Instituto de Quimica, Universidade de Sao Paulo
Caixa Postal 26077, Sao Paulo, Brazil.
Phone: *55-11-815-3810, Fax: *55-11-815-5579,
e-mail henning@iq.usp.br
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Part of this work was published in:
A. Radbruch (ed.): Flow Cytometry and Cell Sorting. Springer Lab Manual. 2 nd edition.
Springer Verlag Berlin, Heidelberg, New York 1999; pp. 140-158. ISBN 3-540-65630-8.
Reprint with permission of the Springer Verlag Berlin, Heidelberg, New York.
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