0 1990 Wiley-Liss, Inc

Cytometry 11:923-927 (1990)

BRIEF REPORT

Improved Method for Measuring Intracellular C a + + With Flu0-3~ Ger T. Rijkers,2 Louis B. Justement, Arjan W. Griffioen, and John C. Cambier Department of Immunology, University Hospital for Children and Youth, “Het Wilhelmina Kinderziekenhuis”, Utrecht, The Netherlands (G.T.R.,A.W.G.); Division of Basic Sciences, Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado (G.T.R.,L.B.J.,J.C.C.) Received for publication February 5, 1990; accepted July 24, 1990

The accuracy of flow cytometric measurement of intracellular calcium with fluo-3 is compromised by variation in basal fluorescence intensity due to heterogeneity in dye uptake or compartmentalization. We have loaded cells simultaneously with fluo-3 and SNARF-1. When SNARF-1 fluorescence is collected at approximately 600 nm, its intensity does notchange upon cell activation. Furthermore, fluo-3 and SNARF-1 fluorescence

With the introduction of calcium-sensitive fluorochromes, measurement of intracellular calcium has become a popular tool for studying early events of cell activation. Out of the various f luorochromes available to date, indo-1 is the fluorochrome of choice for use in flow cytometry (1,7,8). Indo-1 in its acetoxymethylester form can be readily loaded into a variety of cells. Indo-1 exhibits a large shift in its fluorescence emission spectrum upon binding of Ca2+, such that measurement of the ratio of fluorescence intensities a t two wavelengths allows calculation of [Ca2+liindependent of any variability in intracellular dye concentration or instrument efficiency. The use of indo-1 has been limited however since its excitation maximum of 355 nm requires a UV laser or a mercury arc-based flow cytometer. Furthermore, i t must be taken into consideration when working with indo-1 that UV light is potentially injurious to cells and that it precludes the use of “caged’ compounds. Recently, a new calcium-sensitive fluorochrome has been introduced, fluo-3 (43).Fluo-3 is a derivative of fluorescein and its excitation maximum of 488 nm makes it suitable for use on most argon based flow cytometers. Because fluo-3 is analyzed in the visible

signals exhibit a linear relationship. The ratio of fluo-3 to SNARF-1 eliminates a significant proportion of variation in fluorescence intensity caused by variation in fluo-3 uptake and thus can be used as a sensitive parameter for measuring changes in [Ca2+li. Key terms: Fluo-3, SNARF-1, calcium, lympho- cytes

range, problems associated with the use of UV light are avoided. The major disadvantage of fluo-3 is that, upon binding of Ca2+,there is little or no shift in its excitation or emission spectrum, which makes fluo-3 non-ratioable. The fluorescence signal from individual cells is therefore not only dependent on the intracellular calcium concentration but also on cell volume or granularity and intracellular f h o - 3 concentration. Without the ability to ratio the emission or excitation wavelengths of fluo-3, any heterogeneity in dye uptake or compartmentalization, leading to a quantitative variation in the basal fluorescence intensity within a given population of cells, will compromise the accuracy of determinations made for [Ca” ],. Theoretically i t should be

‘This work was supported in part by grant R93-174.89 from the Netherlands Organization for Scientific Research. ‘Address reprint requests to Ger T. Rijkers, Ph.D., Dept. of Immunology, University Hospital for Children and Youth, “Het Wilhelmina Kinderziekenhuis,” P.O. Box 18009, 3501 CA, Utrecht, The Netherlands.

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FIG.1. Dual-parameter display of K46-17 pmX cells loaded with 1 pM fluo-3 (a),0.2 FMSNARF-1 (b), or with fluo-3 and SNARF-1 (c). Cells were analyzed on an Epics 751 flow cytometer equipped with a Coherent argon laser set at 488 nm. F11 was collected at 525 nm, F12 at 610 nm, both with linear amplification.

possible to overcome heterogeneity in dye content by using the ratio of the fluo-3 signal and that of another simultaneously loaded fluorochrome. The assumption must be made that the uptake and intracellular conversion of both fluorochrornes would be equivalent and that the signal of the second fluorochrome would not vary upon cell activation. We have used SNARF-1 (seminaphtorhodafluor) as a fluorochrome for simultaneous loading with fluo-3. SNARF-1, when excited a t 488 nm, shows a pH-dependent emission spectrum between 550 and 650 nm. With increasing pH the emission spectrum shifts to higher wavelengths with a clear pH-independent isoemissive point a t around 610 nm. Because the intracellular pH may change depending on the cell type being studied (6) we have measured SNARF fluorescence a t its isoemissive point. We show here that a ratio of fluo-3iSNARF-1 fluorescence intensities is a more accurate parameter in determination of [Ca2+Iithan the fluorescence intensity of fluo-3 alone.

solved in DMSO (J.T. Baker, Phillipsburg, NJ) a t a concentration of 1 mM and were added directly to cells suspended in buffer A. Cells were incubated a t 37°C for 30 min. Next, a n equal volume of buffer B (IMDM containing 10 mM Hepes, pH 7.4, and 5% FBS) was added and the cells were incubated for a n additional 10 min a t 37°C. Cells were washed two times and resuspended in buffer C (IMDM containing 10 mM Hepes, pH 7.2, 5% FBS, and 10 pgiml DNAse [Sigma]) a t a final concentration of 1 x 106/mL Following loading with fluo-3 and SNARF-1, T celldepleted PBMC were incubated for 30 min a t 4°C with saturating amounts phycoerythrin-conjugated Leu 16 (CD 20; Becton Dickinson), washed, and resuspended in assay buffer (8).

Flow Cytometric Analysis Flow cytometric analysis of [Ca2+Iiwith the Ca2'sensitive dye fluo-3 AM was carried out using a n Epics 751 or FACScan flow cytometer. The Epics 751 (Coulter, Hialeah, FL) was equipped with a 5 W CoMATERIALS AND METHODS herent argon laser (Innova 90-5, Innova, Palo Alto, CA) Cells and a Cicero computer system (Cytomation, EnglePeripheral blood mononuclear cells (PBMC) were wood, CO). The laser excitation wavelength used was isolated by Ficoll Isopaque density gradient centrifu- 488 nm a t 250 mW. Fluorescence emissions were sepgation of heparinized blood from healthy adult donors. arated by a 600 nm dichroic mirror into two-component PBMC were washed and suspended in RPMI-1640 me- emissions which were collected through a 525 nm band dium (Gibco, Grand Island, NY). T cells were depleted pass filter (F11; Fluo-3) and a 610 nm band pass filter by rosetting with 2 aminoethylisothiouronium bro- (F12; SNARF-1). Fluorescence intensity data were colmide-treated sheep red blood cells. K46-17 pmh (a mu- lected during consecutive 5 second increments (-2,500 rine B cell lymphoma cell line, 6) was cultured in Is- cells per increment per channel) and are depicted as a cove's modified Dulbecco's medium (IMDM, Sigma 3-parametric cytogram of fluorescence intensity vs. Chemical Co., St. Louis, MO) supplemented with 10% time vs. cell number. The data from 4 consecutive infetal bovine serum (FBS, Hyclone, Logan, UT) at 37"C, crements was analyzed using the Cicero computer to 8% C02, 100% relative humidity. determine the mean fluorescence intensity. The FACScan flow cytometer (Becton Dickinson) Loading Procedures was used in its standard configuration with a 15 mW, Cells were washed two times and suspended in buffer 488 nm air-cooled argon laser and the standard band A (IMDM, containing 10 mM Hepes, pH 7.0) a t a con- pass filters for F11 (530/30 nm), F12 (585142 nm), and centration of 1 x 106/ml.Fluo-3 AM and SNARF-1 AM F13 (625 nm). Data acquisition was performed using (both from Molecular probes, Eugene, OR) were dis- Chronys or FACScan Research software (both BD);

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Table 1 Increase of [Ca”’], Following Addition of 1 prn Ionomycin to Cells Loaded With F h o - 3 and SNARF-I“ Fluo-3 1 PM 1 PM 1 I*M 1 PM

Loading SNARF-1 0.5 p M

0.2 pM 0.1 p M

F~uo-3 fold increase 2.12 t 0.14 2.08 ? 0.15 2.04 t 0.08 2.32 t 0.17

CV (%) 18.6 18.9 19.5 21.3

Fluo-3ISNARF-1 fold increase 1.83 2 0.09 1.92 2 0.11 2.09 2 0.15

cv (%I 10.5 9.8 11.3

“K46-17 Fmh cells were simultaneously loaded with 1 pM f h o - 3 and various concentrations of SNARF-1. Voltage and gain settings were varied such that the basal fluo-3:SNARF1ratio exhibited a mean channel No. of 75 out of a 250 channel linear scale. Shown are the fold increase in mean fluorescence intensity of fluo-3 and the mean fluo-3:SNARF-1 ratio of 100,000 cells resulting from addition of 1 pM ionomycin (which caused a n increase in [Ca2‘ 1, in 100% of the cells) as well as the coefficient of variation (CV) of basal mean fluo-3 fluorescence intensity and mean fluo-3:SNARF-1 ratio.

data analysis was performed using Chronys, INCA, or Convert software (8).

RESULTS/DISCUSSION K46-17 pmX cells were loaded with 1 pM fluo-3 AM or 0.2 pM SNARF-1 AM or simultaneously with both fluorochromes. Compensation levels on the Epics 751 were set using cells loaded with a single dye so that there was no overlap of the fluo-3 signal in the SNARF-1 channel and vice versa (Fig. la,b). In cells which were loaded with both fluorochromes simultaneously, the fluo-3 and SNARF-1 fluorescence signals were found to exhibit a linear relationship throughout the range of fluorescence intensities (Fig. lc). This supports the premise that cellular uptake and conversion of both fluorochromes is comparable. Next we compared the heterogeneity in the fluo-3/ SNARF-1 ratio signal with that of the fluo-3 signal alone in cells loaded with both fluorochromes simultaneously. In a series of 5 independent experiments, the coefficient of variation (CV) of the fluo-3 signal alone was 17.1 t 1.4, while the fluo-3iSNARF-1 ratio had a CV of 8.5 k 1.2. These data show that using the ratio of fluo-3iSNARF-1 eliminates a significant proportion of the variation in fluorescence intensity which is caused by variation in f h o - 3 uptake. Two experimental approaches were used to test whether this ratio is a useful parameter for monitoring changes in [Ca2 ’ Ii. First, cells which were loaded with 1 pM fluo-3 and various concentrations of SNARF-1 were measured before and after addition of 1 p.M ionomycin. From these data, summarized in Table 1, we can conclude that 5-10 fold lower SNARF-1 concentrations should be used for simultaneous loading with fluo-3: at these concentrations there is no interference with fluo-3 intensity and there is the highest rise in fluo-3iSNARF-1 ratio following addition of ionomycin. Note that the net increase in ratio “intensity” as induced by ionomycin in all cases is slightly lower than the net increase in fluo-3 intensity. However, this decrease is offset by the fact that the fluo-3iSNARF-1 ratio provides greater sensitivity when measuring

small changes in [Ca”], as shown below. Second, the Ca2 ’ response of K46-17 pmX cells to activation by anti-p antibodies was studied. From indo-1 loading experiments it was already known that upon activation with sheep anti-mouse Ig (SAMIG), virtually 100% of K46-17 pmX cells respond with a n increase in ICa2 ’ 1, (data not shown). If however fluo-3 fluorescence intensity is used as a n indicator for [Ca2+],,a significant percentage of the cells apparently does not respond to SAMIG (Fig. 2a). This finding is in agreement with earlier studies in which we showed that fluo-3 is a less sensitive Ca2+ indicator as compared with indo-1 (2). However, if the fluo-3:SNARF-1 ratio rather than the fluo-3 intensity is used to plot the data from the same experiment, all cells are observed to respond following activation with SAMIG (Fig. 2b). We do not know whether activation of K46-17 p.mA cells with SAMIG leads to transient changes in intracellular pH. However, the filter combinations used in the Epics 751 were chosen so t h a t we measured SNARF-1 fluorescence a t its isoemissive point. As shown, SNARF-1 fluorescence intensity (collected a t 610 nm) did not change significantly following activation with SAMIG (Fig. 2c). We have performed similar experiments on a FACScan flow cytometer, a n instrument with a fixed filter set-up. Fluo-3 is measured in the F11 (FITC) channel (525 nm), SNARF-1 in the F13 (TR) channel (625 nm). Figure 3 shows the calcium response of human PBMC activated with concanavalin A (ConA). Also on this instrument, upon simultaneous loading of cells with both f luorochromes, fluo-3 and SNARF-1 fluorescence signals exhibit a linear relationship (Fig. 3a). Following activation with ConA, f iuo-3 fluorescence intensity increases (Fig. 3d), while SNARF-1 intensity remains constant (Fig. 3e). Because the CV of the ratio of fluo3:SNARF-1(28.5 t 2.1) is significantly lower than that of fluo-3 (49.7 i 5.2) and SNARF-1 (35.8 i 4.2), the fluo-3:SNARF-1 ratio (Fig. 3c) is a more sensitive parameter for monitoring changes in [Ca2t ], than the fluo-3 signal (Fig. 3b). With appropriate compensation settings, the FL2

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FIG. 3. Changes in fluo-3 (b,d), fluo-3iSNARF-1 ratio (c,D, and SNARF-1 (el over time in PBMC activated with 10 pgiml ConA (at t = 30 sec). PBMC, loaded with fluo-3 and SNARF-1 were analyzed on a FACScan using FACScan Research software for data acquisition and Convert software for data analysis (a-c). A replicate experiment, using the same cells and identical instrument settings, was performed using Chronys software for data acquisition and analysis (d-0.

(PE) channel (585 nm) on a FACScan can be used for simultaneous cell surface and [Ca2+lianalysis. T celldepleted PBMC were loaded with fluo-3 and SNARF-1 and subsequently cell surface was stained with phycoerythrin (PE)-conjugated anti-CD20 (Leu 16). Cells were run on the FACScan and activated with 10 pgiml F(ab’), fragments of a goat anti-human IgM antiserum (GAHIGM). A two-parameter dot plot of fluo-3 intensity vs. time of the whole cell population shows a small fraction of cells responding upon activation with

GAHIGM (Fig. 4a). The fluo-3 vs. time (Fig. 4c), but more clearly the fluo-3:SNARF-1 ratio vs. time dot plot (Fig. 4d) of B lymphocytes, gated on basis of CD20 expression (Fig. 4b), shows that GAHIGM induces a calcium response in CD20+ B lymphocytes. It appears that simultaneous loading of cells with a second fluorochrome, in addition to fluo-3 is a useful approach to circumvent problems associated with variations in dye uptake. As such, SNARF-1 is a fluorochrome which can be applied provided fluorescence is

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FIG.4. Changes in fluo-3 (a,c), and fluo-YSNARF-1 ratio (d)over time in blood B cells activated with 10 Fgiml F(ab'), fragments of goat anti-human IgM (at t = 3 min). The LCa2 ' 1, response of Leu 16 + cells (26% of total cells; b) is shown in c and d.

collected at approximately 600 nm. Under these conditions the ratio of fluo-3iSNARF-1 is a more sensitive parameter for measuring changes in [Ca2 1, than is fluo-3 fluorescence intensity by itself. +

ACKNOWLEDGMENTS Alex Jansen and Bill Staffopoulos (Becton Dickinson, Erembodegem, Belgium) provided invaluable assistance i n t h e FACScan experiments.

LITERATURE CITED 1. Grynkiewicz G, Poeni M, Tsien RY: A new generation of Ca" indicators with greatly improved fluorescence properties. J Biol Chem 260:3440-3450, 1985. 2. Justement LB, Cambier JC, Rijkers GT, Fittschen C: Use of flow cytometry to study ion transport in lymphocytes and granulocytes.

In: Non-Invasive Techniques in Cell Biology, Grinstein S, Foskett J K (eds). Alan R. Liss, Inc., New York (in press). 3. Justement LB, Reth M, Cambier JC: Membrane IgM and IgD molecules fail to transduce Ca2+mobilizing signals when expressed on differentiated B lineage cells. J Immunol 144:3272-3280, 1990. 4. Kao JPY, Harootunian AT, Tsien RY: Photochemically generated cytosolic calcium pulses and their detection by fluo-3. J Biol Chem 2643179-8184, 1989. 5. Minta A, Kao J P , Tsien RY: Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J Biol Chem 26423171-8178, 1989. 6. Molenaar WH: Effect of growth factor on intracellular pH regulation. Annu Rev Physiol 48:363-376, 1986. 7. Rabinovitch PS, June CS, Ledbetter JA: Heterogeneity among T cells in intracellular free calcium response after mitogenic stimulation with PHA or anti-CD3. Simultaneous use of indo-1 and immunofluorescence with flow cytometry. J Immunol 137:952-961, 1986. 8. Rijkers GT, Griffioen AW, Gregory CD, Keij JF, Zegers BJM: Calcium mobilization in B lymphocytes measured by flow cytometry. Prog Cytometry :63-73, 1989.