Ethan Frome - Purdue University



Detection and purification of rare Ca2+responders by

fixed-time flow cytometry

Attila Tárnok and Henning Ulrich

List of Contents

1. Introduction

2. Materials

3. Procedure

4. Calcium Measurement

5. Fixed Time Device

6. On Line Measurements

7. Results

8. Troubleshooting

9. References

10. Suppliers

11. Figures

___________________________________________________________________________

1. Introduction return

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 time-window 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 return

Cells

( 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 m-chlorophenyl-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 return

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 return

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 μm 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 calcium-free buffer containing 10 mM HEPES, pH 7.2, 5 mM sodium azide, 5 mM deoxy-D-glucose, 20 µM CCCP, 5 µg/ml nigericin, 4 µΜ ionomycin, and 5 µΜ 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 return

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 return

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 return

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 HA-induced 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 non-responsive 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 return

( 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 non-responsiveness 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 100-1,000 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 ()

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 return

• 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. return

• 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 flow-cytometry. 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 return

Suppliers Product

American Type Culture Selection (ATCC) P19 rat embryonal carcinoma

12301 Parklawn Drive cells

Rockville, MS 20582-1776, U.S.A.



Bachem, Feinchemikalien AG Head activator (customer

Hauptstr. 144, Bubendorf CH-4416, desired synthesis)

Switzerland,

Phone and Fax: +41-61-9312333

Bückmann GmbH, Technische Gewebe Nylon gauze of 50 μM 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 DMEM medium, antibiotics,

Box 6009 glutamate, sodium pyruvate,

Gaithersburg, MD 20884-9980, U.S.A. non-essential amino acids

Phone: +1-301-840-8000

Fax +1-301-258-8238



Molecular Probes, Indo-1 AM, pluronic F-127,

PO Box 22010, Eugene, OR 97402-0469, U.S.A. propidium iodide

Phone: +1-541-465-8300

Fax: +1-541-344-6504

E-mail: tech@

Omega Optical optical filters, dichroic mirrors

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.

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 dichroic mirror,

Box 2480 optical filters

D-55014 Mainz

Fax: +49-61 31-66 20 00

Phone: +49-61 31-66-0



Sigma Chemical Company insulin, transferrin, sodium

Box 14508 selenite, retinoic acid,

St. Louis, Missouri 63178-9916, U.S.A. HEPES, Br-A 23187, sodium

Phone: +1-314-771-5750 azide, 2-deoxy-D-glucose,

Fax: +1-314-771-5757 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@

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@

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 DAS software package

GSF-AG Durchflusszytometrie

Ingolstädter Landstr. 1

D-85764 Neuherberg Germany

E-mail: Beisker@gsf.de

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 return

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 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. 2nd 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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