ARTICLE |
Correspondence to: Brigitte Vandewalle, UPRES 1048/ERITMINSERM, Université de Lille, 1 Place de Verdun, 59045 Lille, France. E-mail: bvandewalle@univ-lille2.fr
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Summary |
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Pancreatic ß-cells contain large amounts of zinc. We took advantage of this to try to localize, quantify, and isolate insulin-producing cells from islet preparations. Our study was designed to identify a non-toxic zinc-sensitive fluorescent probe able to selectively label labile zinc in viable ß-cells and to exhibit excitation and emission wavelengths in the visible spectrum, making this technique exploitable by most instruments. We tested Newport Green, a probe excitable at 485 nm with a dissociation constant in the micromolar range corresponding to a low affinity for zinc. The loading of the lipophilic esterified form of Newport Green was easy, rapid, specific, and non-toxic to cells. Confocal microscopy highlighted an intense fluorescence associated with secretory granules. Regression analyses showed a good relationship between zinc fluorescence and islet number (r=0.98) and between zinc fluorescence and insulin content (r=0.81). The determination of Zn fluorescence per DNA enabled us to assess the quality of the different islet preparations intended for islet allografting in terms of both purity and viability. Cell sorting of dissociated Newport Green-labeled cells resulted in a clear separation of ß-cells, as judged by insulin content per DNA and immunocytochemical analysis. This zinc probe, the first able to specifically label living cells in the visible spectrum, appears very promising for ß-cell experimentation, both clinically and for basic research. (J Histochem Cytochem 49:519527, 2001)
Key Words: human pancreatic ß-cell identification, human pancreatic islet purification, fluorescence-activated cell sorting, zinc-sensitive fluorescent probe
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Introduction |
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PANCREATIC ISLETS contain a much higher concentration of zinc than other tissues. Zinc plays an important role in packaging insulin because it is firmly established as an integral part of the insulin crystal as a 2-Zn-insulin hexamer. In addition, free ionized zinc is found in the extragranular space of ß-cells, where it may act as a reservoir for granular zinc (
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Materials and Methods |
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Human Islet Processing
Human pancreata (n=11; mean age 35 ± 12 years) were harvested from adult brain-dead donors in accord with French Regulations and with the local Institutional Ethical Committee. Pancreatic islets were isolated after ductal distension of the pancreata and digestion of the tissue with Liberase (Roche Diagnostics; Meylan, France) according to the automated method of
Cell Culture
Pancreatic ß-cells of murine origin, including INS-1 kindly obtained from Dr. Wolheim (
Fluorescence Labeling with Newport Green
Newport Green (Molecular Probes Europe; Leiden, The Netherlands) is available as a salt (potassium form, NG-K) or an ester (diacetate form, NG-Ac). The water-soluble potassium form was used for fluorometric studies designed to determine both binding constants from titration curves of ZnCl2 and specificity from other metal ion solutions. NG-K (6 µM) was added to 96-well microplates and left in the dark for 10 min. Fluorescence was measured at room temperature in a fluorophotometer equipped with microtiter plate accessories (Fluorocount; Packard, Rungis, France). The Newport Green indicator exhibited an increase in fluorescence emission on binding Zn, with a slight shift in wavelength; single excitation and emission spectral peaks were measured at wavelengths of 485 and 530 nm, respectively.
The ester form (NG-Ac) was used for fluorescent studies on living cells. NG-Ac is cell-permeant and can therefore diffuse across cell membranes. Once inside the cells, this ester is cleaved by intracellular esterases to yield a cell-impermeant fluorescent indicator able to bind zinc. Cells in PBS were exposed for 30 min at 37C to 25 µM NG-Ac containing 1.5 µl/ml Pluronic F127 to enhance the penetration of the probe. After washing in PBS, the fluorescence was estimated in microwell plates as described above or analyzed by confocal microscopy or cell sorting. Standardization of fluorescence in spectrofluorometric measures was achieved using a blank consisting of 25 µM NG-Ac in PBS set at a fixed and constant unit of fluorescence.
Cell Distension and Dissociation
Distension of islets was performed for 5 min at 37C in a dispersion buffer consisting of Earle's balanced salt solution without Ca2+ and Mg2+ and supplemented with 10 mM HEPES, 3 mM EGTA, 2.8 mM glucose, and 2.5 mg/ml BSA.
Total dissociation of cells from islet preparations was achieved on islet pellets (200 x g, 3 min) by gentle pipetting for 10 min in 4.5 ml dispersion buffer. An enzymatic dissociation was then performed by the addition of 100 µg/ml trypsin. The reaction was stopped by 2 volumes FCS and 1 volume 0.035% (w/v) trypsin inhibitor (Type 1-S; Sigma) when about 80% of islet cells appeared as single cells. The cell suspension was centrifuged (500 x g, 5 min) and the pellet suspended in F10 medium without Ca2+ and containing 2.8 mM glucose. The cell suspension was then filtered through a 70-µm nylon screen.
Confocal Microscopy
Confocal microscopic analyses were performed using a confocal inverted microcope (Leica TCS-NT; RueilMalmaison, France) on dissociated cells or on whole islets plated in Lab-Tek chambers with coverslips (Nalge Nunc International; Naperville, IL) coated with poly-L-lysine. When islets were particularly compact, we performed a mild dissociation of islets before staining for 5 min in the dispersion buffer to prevent defective penetration of the probe. Staining was performed after a period of 90 min in F10 medium containing 2.8 mM glucose to restore cell integrity.
Cell Sorting
Cell sorting was performed after total dissociation of islet cells. Staining was achieved after a period of 90 min in F10 medium without Ca2+ and containing 2.8 mM glucose.
We used either an Epics XL-MCL or an Epics Elite Coulter flow cytometer equipped with an argon-ion laser (Coultronics; Margency, France). Excitation was performed with the 488-nm blue line of the laser and emission measured through a 530/30 bandpass filter.
Insulin Content and Secretion
Insulin content in human pancreatic islets was measured after cell pelleting by centrifugation and homogenization by sonication (20 Khz, 30 W) for 15 sec in 200 µl Tris-HCl containing 1 mM EDTA, pH 7.4, and frozen-stored at -20C. The islet insulin content was measured by radioimmunometric assay (Bi-insulin; IRMA Diagnostics, Pasteur, France) after an overnight acidethanol (0.18 M HCl in 95% ethanol) extraction of 50-µl aliquots of the homogenates at 4C. Insulin secretory responses were studied as follows: 1 hr preincubation in Kreb's buffer containing 3.6 mM glucose followed by three successive incubation periods of 1 hr each with 3.6, 16.7, and 3.6 mM glucose. Incubation media were collected for assay of insulin secretion from the cells. The stimulation indices were calculated by dividing insulin secretion in the presence of high glucose by mean basal insulin secretion levels.
DNA Assay
DNA was assayed in aliquots of homogenates using Pico Green (Molecular Probes) as the fluorescent probe at a 1:200 dilution in PBS containing 20 mM EGTA. After a brief period of contact (15 min), the samples were excited at 485 nm and the fluorescence emission intensity measured at 530 nm.
Immunocytochemical Analysis
Cells were cultured on glass coverslips, washed with PBS to remove serum, and processed at 4C as described by
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Results |
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Properties of Newport Green
Fluorescence of NG-K (6 µM) in 10 mM Tris-HCl was increased at the micromolar range of Zn2+ in the form of ZnCl2 or crystal insulin (Actrapid) and was saturated at about 10 µM Zn2+. Half-maximal intensity was achieved at a concentration of 23 µM Zn2+. Binding studies using NG-Ac confirmed that the ester form was unable to bind zinc (Fig 1).
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Zinc-dependent fluorescence of NG-K was unaffected by an excess of the metal ions frequently encountered in living cells, such as Ca2+, Mg2+, or Fe2+ added as salts. Among other metal ions analyzed, Cu2+, Cd2+, and Pb2+ quenched fluorescence, whereas others, such as Co2+ and Ni2+ enhanced Zn fluorescence. Nevertheless, they can be found only as trace elements in living cells. As expected, EDTA, EGTA, and an acidic pH affected Zn fluorescence of NG-K (Fig 2).
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Control of Cell Loading
"Hand-picked" islets loaded with 25 µM NG-Ac demonstrated an excellent correlation between fluorescence intensity and islet number (r=0.99; p< 0.0001) (Fig 3).
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Fluorescence intensity was also increased in murine cells cultured in the presence of ZnCl2 (25 µM) and pyrithione (4 µM), a zinc ionophore. The ratios of Zn:DNA in arbitrary units were 2.5- and 12-fold higher in treated INS-1 and in ß-TC3, respectively, compared to the control cells.
Toxicity Study
INS-1 cells stained with NG-Ac (25 µM) for 30 min and then cultured for 7 days exhibited the same pattern of growth as untreated cells (Fig 4).
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As shown in Table 1, human pancreatic islets stained by NG-Ac (25 µM, 30 min) demonstrated the same capacity for glucose-induced insulin secretion as control islets. In contrast, DTZ staining (30 min, 10 µg/ml) resulted in a moderate shift of stimulatory indices.
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Cell Imaging
Islet cell staining was visualized by confocal microscopic studies. Cells positively stained by NG appeared green; dead cells stained by propidium iodide (4 µM) appeared red (Fig 5A and Fig 5B). Analyses of individual optical sections of islets indicated a homogeneous staining from the periphery to the center in cases where cells were loosely bound inside the islet (Fig 5D). However, defective penetration of the probe was noticed when islets were particularly compact (Fig 5C). We therefore systematically performed a mild distension of islets for 5 min, as described above, before staining.
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Staining of individual cells revealed a heterogeneous intensity of fluorescence with cytoplasmic dots (Fig 5E). Identical pictures were obtained using insulin staining by fluorescein-conjugated antibody to insulin/proinsulin. No nuclear staining was noticed (Fig 5F).
Quantitative Studies
FACS analyses of NG-stained cells from semipurified preparations (containing endocrine ß- and non-ß-cells contaminated with exocrine cells) demonstrated a large range (about x300) of relative fluorescence units (RFUs). In comparison, the range of autofluorescence was more limited (about x10) (Fig 6). The recovery of cells after sorting was always greater than 85%, and viability assessed by trypan blue exclusion was greater than 95%.
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Two populations of cells were observed, according to their side scatter patterns (SS) and RFU, and were gated for sorting (Fig 7). The ratios of mean values (±SEM) in the two populations were 10.5 ± 1.8 (n=5) for SS and 12.1 ± 1.14 (n=5) for RFU. Insulin content of the sorted cells was analyzed. The ratios of insulin per DNA in the two sorted populations were 30 ± 9.4 (n=5), proving a clear distinction between ß- and non-ß-cells. Immunocytochemical analysis of these two sorted cell populations also confirmed the presence of a great majority of insulin-positive cells (red staining) in the most fluorescent sorted population compared to the less fluorescent one (Fig 8). When the cell populations were compared before sorting, therefore containing endocrine plus exocrine cells, vs the most fluorescent sorted population, the enrichment in ß-cells, estimated by the ratios of insulin per DNA, was highly variable (from 1.3 to 7; mean 3.2 ± 1.04; n=5) because it depended on the purity of the starting preparation, i.e., the more pure the starting preparation was, the less the enrichment ratio was.
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In terms of function, the dissociated cells plated before and after sorting and cultured for 72 hr exhibited the same pattern of glucose-induced insulin secretion. However, the most fluorescent sorted cells secreted about 10-fold more insulin than the weakest fluorescent cells (Table 2).
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The determination of zinc content per DNA unit was investigated in islet preparations from different donors to determine if an index designed to estimate the purity plus viability of these preparations could be established. Staining of cells was performed in six replicates of 40 IEs stained with 25 µM NG-Ac after mild distension of islets. As shown in Fig 9, we observed a wide variability of values among the different preparations. Regression analyses demonstrated fair correlations between Zn:DNA values and purity, qualitatively estimated by the percentage of DTZ-stained cells in the preparation (r=0.63; p=0.011, n=15) and between Zn:DNA values and insulin content (r=0.81; p=0.007; n=9).
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Discussion |
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Pancreatic ß-cells contain large amounts of zinc used in synthesis, storage, and secretion of insulin. We therefore screened, among the available fluorescent probes, those able to fulfill two criteria: to selectively label labile zinc in viable cells and to exhibit excitation and emission wavelengths in the visible spectrum so as to be exploitable by most instruments, particularly cell sorters and the confocal microscope.
Among the divalent chelating stains used for islet microscopic examination in vitro, dithizone (DTZ), the most popular, produces bright red islets when viewed by white-light microscopy. DTZ may also produce fluorescence when dissolved in DMSO; however, its fluorescence fades too quickly for reliable sorting. Other probes have been used for staining cellular labile zinc, including quinoline derivatives such as TSQ (N-[6-methoxy-8-quinolyl]-para-toluenesulfonamide (-methyl-8-p-toluenesulfonamido-6-quinolyloxy acetic acid) (
Here we tested a recently identified potential Zn2+ indicator, Newport Green (NG) (
Loading of the lipophilic esterified form of Newport Green was easy, rapid, and non-toxic to cells. Examination of its distribution inside the cells by confocal microscopy highlighted an intense NG fluorescence mainly associated with secretory granules, demonstrating that NG competed with insulin for zinc. Moreover, the nucleus was not stained by NG.
Analysis of serial optical sections from confocal microscopic examination revealed a lack of penetration of the probe in the core of the islets when they were particularly big and compact. This drawback could be avoided by systematic mild distension of islet cells before staining. Under these conditions, a linear relationship between fluorescence intensity and islet number could be spectrofluorometrically demonstrated.
FACS analysis of semipurified preparations of islet cells revealed a great range of fluorescence intensities, which were gated for sorting according to the side-scattering (SS) activity of cells. Two populations of particles were isolated, one with the larger light-scattering properties and NG fluorescence corresponding to insulin-containing cells, and the other with smaller values of the two parameters corresponding to non-ß-cells. ß-cells displayed about a 12-fold higher NG fluorescence than non-ß-cells, and their light scatter activity was also 10-fold higher. As a result, a clear distinction between the two populations could be easily achieved for research applications. Moreover, the sorted insulin-containing cells maintained their function in terms of glucose-induced insulin secretion.
For clinical purposes, pancreatic islet transplantation is now recognized as a treatment of choice for patients with Type 1 diabetes mellitus (
As a whole, Zn staining by NG appears to be a valuable method to specifically identify, quantify, and isolate insulin-secreting cells from semipurified preparations of human pancreatic islet cells. This Zn probe, the first able to specifically label living cells in the visible spectrum, therefore appears very promising in ß-cell handling for both clinical and research applications.
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Acknowledgments |
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Supported by grants from INSERM CRMD413.
We are indebted to Dr M. Labalette and Ms C. Grutzmacher (Laboratoire d'Immunologie, Faculté de Médecine Lille) for their expertise in cell sorting. We thank the "Service Commun d'Imagerie Cellulaire: IFR 22" for access to the confocal microscope and the fluorophotometer.
Received for publication December 6, 2000; accepted December 6, 2000.
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