1 Department of Medicine, 2 The Howard Hughes Medical Institute, 3 Departments of Pathology, 4 Neurobiology, Pharmacology and Physiology, and 5 Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637; and 6 Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have generated transgenic mice
that express green fluorescent protein (GFP) under the control of the
mouse insulin I gene promoter (MIP). The MIP-GFP mice develop normally
and are indistinguishable from control animals with respect to glucose
tolerance and pancreatic insulin content. Histological studies showed
that the MIP-GFP mice had normal islet architecture with coexpression
of insulin and GFP in the -cells of all islets. We observed GFP
expression in islets from embryonic day E13.5 through adulthood.
Studies of
-cell function revealed no difference in glucose-induced
intracellular calcium mobilization between islets from transgenic and
control animals. We prepared single-cell suspensions from both isolated islets and whole pancreas from MIP-GFP-transgenic mice and sorted the
-cells by fluorescence-activated cell sorting based on their green
fluorescence. These studies showed that 2.4 ± 0.2%
(n = 6) of the cells in the pancreas of newborn (P1)
and 0.9 ± 0.1% (n = 5) of 8-wk-old mice were
-cells. The MIP-GFP-transgenic mouse may be a useful tool for
studying
-cell biology in normal and diabetic animals.
insulin; diabetes
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE INSULIN-PRODUCING
-CELL of the pancreas plays a central role in the
pathophysiology of diabetes mellitus, with anatomic and functional loss
of these cells leading to type 1 and type 2 diabetes mellitus,
respectively (2). The identification and characterization
of embryonic or adult stem cells that give rise to the
-cell could
lead to cellular-based therapies for treating both forms of diabetes
(1, 18, 22). Previous studies have shown that green
fluorescent protein (GFP) from the jellyfish Aequorea
victoria and its yellow and cyan derivatives could be utilized as
reporter genes to label specific cell types including pancreatic
-cells by expressing GFP under the control of a tissue-specific promoter (5, 6, 8-10, 15, 23, 28). One advantage of these proteins is that they can be detected in living cells because they fluoresce brightly upon exposure to ultraviolet light or blue
light without the addition of coactivators or exogenous substrate. In
addition, pure populations of fluorescent-tagged cells can be isolated
using a fluorescence-activated cell sorter (FACS) (10, 15,
23). Rat and human
-cells treated with recombinant adenovirus
expressing green fluorescent protein (GFP) under the control of the rat
insulin I promoter appear to function normally, suggesting that
expression of GFP may be well tolerated by these cells (9,
23). Thus we reasoned that, if we could generate a mouse model
in which we had genetically tagged the pancreatic
-cells with GFP,
we would have a potentially valuable research tool for studying
-cell biology, including the identification of progenitor cells.
Here, we describe a line of transgenic mice in which the pancreatic
-cells are genetically tagged with GFP.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of mouse insulin I gene promoter-GFP-transgenic mice.
The mouse insulin I gene promoter (MIP)-GFP-transgenic construct was
assembled using an 8.5-kb fragment of the MIP that includes a region
from 8.5 to +12 bp (relative to the transcriptional start site), the
coding region of enhanced GFP (EGFP) (0.76 kb; Clontech, Palo Alto,
CA), and a 2.1-kb fragment of the human growth hormone (hGH) cassette
gene for high-level expression (25, 26). The 11.2-kb
MIP-EGFP-hGH fragment was isolated from the vector by digestion of the
plasmid construct with SfiI and HindIII and agarose gel electrophoresis. The fragment was further purified using an
Elutip-D column (Schleicher & Schuell, Keene, NH). The purified
transgene DNA was microinjected into the pronuclei of CD-1 mice by the
Transgenic Mouse/ES Core Facility of the University of Chicago Diabetes
Research and Training Center (DRTC). Tail DNA from potential founder
mice was screened for the presence of the transgene by PCR using
forward and reverse primers 5'-GAAGACAATAGCAGGCATGCTG-3' and
5'-ACTGGGCTTACATGGCGATACTC-3', respectively.
Glucose tolerance testing. Intraperitoneal glucose tolerance tests (IPGTTs) were performed after a 4-h fast. Blood was sampled from the tail vein before and 30, 60, 90, and 120 min after intraperitoneal injection of 2 mg/g body wt of dextrose. Glucose levels were measured using a Precision Q.I.D. Glucometer (MediSense, Waltham, MA).
Insulin assays. Pancreatic insulin content was measured after acid ethanol extraction of the whole pancreas, as described previously (24). Insulin concentration was measured by a double-antibody radioimmunoassay using a rat insulin standard in the Radioimmunoassay Core Laboratory of the University of Chicago DRTC. The intra-assay coefficient of variation for this assay is 7%. All samples were assayed in duplicate.
Isolation of pancreatic islets of Langerhans. Pancreatic islets were isolated using a modification of the procedure originally described by Lacy and Kostianovsky (21). Briefly, the pancreas was inflated with a solution containing 0.3 mg/ml collagenase (Type XI; Sigma, St. Louis, MO) in Hanks' balanced salt solution, injected via the pancreatic duct. The inflated pancreas was removed, incubated at 37°C for 10 min, and shaken vigorously to disrupt the tissue. After differential centrifugation through a Ficoll gradient to separate islets from acinar tissue, the islets were washed and then hand picked. They were plated on 12- or 35-mm coverslips to facilitate adherence for subsequent measurement of intracellular calcium or confocal microscopic visualization. The islets were cultured in RPMI 1640 supplemented with 10% (vol/vol) fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified incubator at 37°C in 95% air and 5% CO2.
Preparation of single-cell suspensions from islets and pancreas. Isolated islets were incubated in a solution of 0.05% trypsin-EDTA (GIBCO, Grand Island, NY) at 37°C for 3 min. The digestion was stopped by adding RPMI 1640 with 10% (vol/vol) FBS. The pancreata from newborn (P1) mice were removed and digested in a solution containing 0.3 mg/ml collagenase and then in 0.05% trypsin-EDTA. The resulting single cells were washed with PBS, resuspended in cold PBS with 10% (vol/vol) FBS, and filtered using 70-µm mesh. Cells were stained with trypan blue to check viability, and preparations showing >95% live cells were analyzed. The pancreatic cell suspension was diluted to 2 × 106 cells/ml with the PBS-FBS solution and then fixed in 4% paraformaldehyde.
Pancreatic islet morphology.
The pancreas was removed, embedded in optimum cutting temperature
compound (Tissue-Tek O.T.C., Sakura Finetek, Torrance, CA) and frozen
in isopentane at 70°C. Serial sections were cut at 6 µm in
thickness and fixed in 4% paraformaldehyde. GFP fluorescence is well
retained under these conditions (24). The sections were stained with a polyclonal guinea pig anti-porcine insulin antibody to
identify
-cells and with polyclonal rabbit anti-human glucagon, somatostatin, and pancreatic polypeptide antibodies to identify
-,
-, and PP cells, respectively (DAKO, Carpinteria, CA). Sections were
also stained with a monoclonal anti-mouse GFP antibody (Clontech) to
detect GFP expression and with a polyclonal goat anti-hGH antibody (DAKO) to test for expression of GH from the transgene construct. The
primary antibodies were detected using Texas red-conjugated anti-guinea
pig/rabbit/goat IgG (H+L) and biotin-streptavidin-conjugated anti-mouse
IgG (H+L) followed by Texas red-conjugated streptavidin (Jackson
ImmunoResearch Laboratory, West Grove, PA). Microscopic images were
taken with a Nikon Eclipse E800 microscope with PCM-2000 (Nikon, New
York, NY) and an Olympus SZX-RFL3 microscope (Olympus, Melville, NY).
Intracellular calcium measurements.
The GFP expression interferes with fura 2 signals, and as a
consequence, the calcium indicator fura 2 cannot be used to monitor changes in intracellular calcium in GFP-expressing -cells. The short-wavelength excitation of the EGFP excitation spectrum extends down to ~350 nm, which is in the range of fura 2. Thus excitation of
fura 2 will excite EGFP and contaminate the common emission spectrum.
Fura red is a calcium indicator with a large stokes shift, thereby
minimizing the contamination of the calcium signal by GFP (20,
30). Fura red has been successfully used in combination with GFP
expression in other studies (11, 12); however, there are
reports of some signal cross talk when used with EGFP (3). Another calcium indicator, X-rhod-1, which has excitation/emission maxima of ~580/602 nm, may also give a good separation of the signals
from GFP and the calcium indicator (3); however, care must
be taken, since X-rhod-1 has a certain selectivity for mitochondria (13). We used fura red in the studies described here. The
isolated islets were loaded with 5 µmol/l fura red-AM (acetoxymethyl
ester; Molecular Probes, Eugene, OR), and changes in intracellular
calcium were monitored using a Fluoview scanning laser confocal lens
with an inverted IX70 microscope (Olympus). Tiempo real-time
acquisition software (Olympus) was used to collect and plot the data.
The Cy5 filter set (700/75-nm bandpass filter set) was used to detect fura red signals and to eliminate the contamination with GFP signals. Solutions were perfused in a temperature-controlled chamber by use of a
TC-344 Dual Heater Controller (Warner Instrument, Hamden, CT). All the
measurements were performed at 34-35°C.
Flow cytometric analysis.
Flow cytometric analysis of GFP-labeled -cells was carried out using
a FACScan flow cytometer with Cell Quest software (Becton Dickinson,
Franklin Lakes, NJ). GFP-expressing cells were detected using the FL1
channel (absorption spectra 530/30 nm). We have sorted both fixed and
nonfixed cells. However, the studies described here were carried out
using cells fixed as described above.
Statistical analysis. Results are expressed as means ± SE. We compared groups by use of ANOVA (StatView software; SAS Institute, Cary, NC). Differences were considered to be significant at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of GFP in -cells.
The MIP-GFP construct was injected into the fertilized eggs of CD-1
mice. We obtained three founders: one female (6504) and two males (6502 and 6508). GFP was expressed in the islets of all three founders. The
founders 6502 and 6508 were estimated to have one copy of the transgene
by quantitative real-time PCR. The founder 6504 was estimated to have
five copies, and two of her male F1 pups, 6729 and 6719, each had one
copy of the transgene. Progeny from transgenic lines 6502 and 6719 showed delayed recovery on IPGTT and nonuniform expression of GFP
within their islets and were not studied further. The studies described
here were carried out on line 6729 [Tg(MIP-GFP)6729Hara]. The
transgene has been maintained on the CD-1 background, and mice have
been housed under specific pathogen-free conditions with free access to
food and water.
|
Physiological characterization of MIP-GFP mice.
The MIP-GFP mice developed normally. At 6 wk of age, there were no
significant differences in body weight, fasting blood glucose, and
pancreatic insulin content between transgenic and nontransgenic CD-1
male mice (data not shown). IPGTT at 6 wk showed no statistically significant difference in the response between transgenic and nontransgenic male animals (Fig. 2). We
followed body weight and performed IPGTTs on the transgenic mice up to
40 wk and body weight and nonfasting blood glucose levels up to 60 wk,
and none of the animals has shown any evidence of abnormal weight or
hypo- or hyperglycemia (data not shown.)
|
Glucose responsiveness of pancreatic islets from the MIP-GFP mice.
We examined the effects of glucose on intracellular calcium
mobilization in the islets of the MIP-GFP mice as a measure of -cell
function. We used fura red (16) to monitor intracellular calcium levels to allow measurement of signals from the calcium indicator in the presence of GFP. The islets from both MIP-GFP and
nontransgenic mice exhibited a robust mobilization of calcium in
response to 10 mM glucose, with no apparent difference between the
islets from transgenic and nontransgenic animals (Fig.
3). Similar responses were observed in
response to 20 mM glucose and 50 mM KCl, occasionally accompanied by
oscillations in calcium levels (data not shown).
|
FACS analysis of GFP-labeled -cells.
The islets isolated from MIP-GFP mice (8 wk old) were dissociated into
single cells. The GFP-labeled
-cells could be readily separated from
the non-
-cells cells by FACS (Fig.
4 shows a representative trace).
|
Measurement of pancreatic -cell number.
A single-cell suspension was prepared from whole pancreas of a
MIP-GFP-transgenic mouse at P1 (Fig.
5A), and the percentage of
pancreatic
-cells was measured by FACS. Flow cytometric analysis revealed that 2.6% of the pancreatic cells expressed GFP (Fig. 5B). Further analyses on five additional transgenic
littermates gave similar results and indicate that 2.4 ± 0.2%
(n = 6) of the cells in the P1 pancreas were
GFP-expressing
-cells (each trace not shown). We also prepared
single-cell suspensions from whole pancreas of older mice and sorted
the cells on the basis of GFP expression. We found that 0.9 ± 0.1% (n = 5) of the cells in pancreas of 8-wk-old
animals were GFP positive (data not shown).
|
Expression of MIP-GFP transgene during development.
The pancreas begins to form at the 26-somite stage (E9.5) of gestation
in the mouse (14). Although insulin mRNA can be detected at the 20-somite stage (14), significant expression of
insulin begins only at E13.5 (17). We observed GFP-labeled
cells in the pancreas at E13.5 (we did not examine earlier stages) and continuing throughout life (Fig. 6). At E13.5, there is a scattered mass of GFP-expressing cells in the pancreas adjacent to the duodenum. GFP-labeled islets are evident at E18.5 and in the adult (60 wk), where
they can be readily seen distributed throughout the pancreas.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have generated transgenic mice in which pancreatic -cells
are genetically tagged with GFP. The phenotypic characterization of the
MIP-GFP transgenic mice suggests that the presence of cytoplasmic GFP
does not impair
-cell development or function, at least in the line
of mice described in this report. We can envisage a number of uses of
these mice for studying
-cells in situ and in isolation. In this
regard,
-cell function can be studied in the MIP-GFP mice or in
intercrosses with other genetically engineered and mutant mice. We
believe that the MIP-GFP mice will be useful for isolating pancreatic
-cells at various stages of development from embryonic to adult. The
purified
-cells can be used for molecular biological studies such as
monitoring the changing pattern of gene expression during development
with the use of microarrays (27) or for biophysical
studies of their functional properties (19). The isolation
of a pure population of
-cells is arduous. Van de Winkel et al.
(29) have described a procedure that works well with rat
islets, beginning with purified islets. The islets are disassociated
into single cells, from which a highly enriched
-cell population can
be obtained by FACS on the basis of the high intrinsic autofluorescence
of
-cells. Meyer et al. (23) have described another
procedure, in which dispersed islet cells (in this case human) were
infected with a recombinant adenovirus expressing GFP driven by the rat
insulin I promoter. The GFP is expressed only in
-cells, and these
cells can be sorted by FACS to obtain a pure (>95%) population of
-cells. This procedure is generally applicable to isolating
-cells from many different species, including mice, and at various
stages of development. However, special precautions must be taken when
working with adenovirus, even the attenuated vectors that are commonly
used in molecular biology. In contrast to these procedures, purified
-cells can be readily isolated from MIP-GFP mice, beginning with
either islets or pancreas. The ability to flow sort disassociated
pancreatic cells to isolate them will facilitate studies of embryonic
-cells when islet isolation is very difficult. A second use of the
MIP-GFP-transgenic mice is in studies where real-time instant
identification of
-cells is required, such as in
electrophysiological studies, as the
-cells can be distinguished
from non-
-cells on the basis of their green fluorescence. Another
use is in studies of
-cell development.
-cell progenitors and/or
stem cells have been described in ductal tissue (4), adult
marrow (18), and embryonic stem cells (1, 22), and the MIP-GFP mice may be useful in identifying the
progenitor/stem cells in these tissues and cells. The preliminary data
that we have presented here indicate that we can use FACS to quantify the number of
-cells in the pancreas, and this may be a useful method for following the changes in
-cell number that occur in different physiological states such as pregnancy and diabetes. We also
believe that we may be able to use FACS analysis to monitor changes in
-cell size. Finally, the GFP fluorescence is sufficiently intense,
especially in the adult, to be detected within a thick specimen,
including a whole pancreas (Fig. 6).
Thus it may be possible to carry out a three-dimensional
reconstruction of the distribution of islets within the entire pancreas
of the MIP-GFP mice.
In summary, we have generated a line of mice in which the -cells are
genetically tagged with GFP. We believe that the MIP-GFP mice will be a
useful tool for studying
-cells and islets in normal and diabetic states.
![]() |
ACKNOWLEDGEMENTS |
---|
This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-20595, DK-44840, and DK-61245. M. Hara is a Naomi Berrie Fellow, and G. I. Bell is an Investigator of the Howard Hughes Medical Institute.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: M. Hara, Howard Hughes Medical Institute Research Laboratories, The Univ. of Chicago, 5841 South Maryland Ave., MC1028, Chicago, IL 60637 (E-mail: mhara{at}midway.uchicago.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
September 17, 2002;10.1152/ajpendo.00321.2002
Received 17 July 2002; accepted in final form 10 September 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Assady, S,
Maor G,
Amit M,
Itskovitz-Eldor J,
Skorecki KL,
and
Tzukerman M.
Insulin production by human embryonic stem cells.
Diabetes
50:
1691-1697,
2001
2.
Bell, GI,
and
Polonsky KS.
Diabetes mellitus and genetically programmed defects in -cell function.
Nature
414:
788-791,
2001[ISI][Medline].
3.
Bolsover, S,
Ibrahim O,
O'Luanaigh N,
Williams H,
and
Cockcroft S.
Use of fluorescent Ca2+ dyes with green fluorescent protein and its variants: problems and solutions.
Biochem J
356:
345-352,
2001[ISI][Medline].
4.
Bonner-Weir, S,
Taneja M,
Weir GC,
Tatarkiewicz K,
Song KH,
Sharma A,
and
O'Neil JJ.
In vitro cultivation of human islets from expanded ductal tissue.
Proc Natl Acad Sci USA
97:
7999-8004,
2000
5.
Chalfie, M,
and
Kain S
(Editors).
GFP Green Fluorescent Protein: Properties, Applications, and Protocols. New York: Wiley-Liss, 1996.
6.
Chalfie, M,
Tu Y,
Euskirchen G,
Ward WW,
and
Prasher DC.
Green fluorescent protein as a marker for gene expression.
Science
263:
802-805,
1994[ISI][Medline].
7.
Clontech Laboratories
Living Colors User Manual. Palo Alto, CA: Clontech, 1997. (PT2040-1).
8.
Conn, PM
(Editor).
Methods in Enzymology 302: Green Fluorescent Protein. New York: Academic, 1999.
9.
De Vargas, LM,
Sobolewski J,
Siegel R,
and
Moss LG.
Individual cells within the intact islet differentially respond to glucose.
J Biol Chem
272:
26573-26577,
1997
10.
Diamond, RA,
and
DeMaggio S
(Editors).
In Living Color: Protocols in Flow Cytometry and Cell Sorting. New York: Springer Verlag, 1999, p. 199-226.
11.
Doherty, AJ,
Coutinho V,
Collingridge GL,
and
Henley JM.
Rapid internalization and surface expression of a functional, fluorescently tagged G-protein-coupled glutamate receptor.
Biochem J
341:
415-422,
1999[ISI][Medline].
12.
Edwards, BS,
Kuckuck FW,
Prossnitz ER,
Okun A,
Ransom JT,
and
Sklar LA.
Plug flow cytometry extends analytical capabilities in cell adhesion and receptor pharmacology.
Cytometry
43:
211-216,
2001[ISI][Medline].
13.
Gerencsér
ÁA and Adam-Vizi V. Selective high-resolution fluorescence imaging of mitochondrial Ca2+ concentration.
Cell Calcium
30:
311-321,
2001[ISI][Medline].
14.
Gittes, GK,
and
Rutter WJ.
Onset of cell-specific gene expression in the developing mouse pancreas.
Proc Natl Acad Sci USA
89:
1128-1132,
1992[Abstract].
15.
Hadjantonakis, AK,
and
Nagy A.
FACS for the isolation of individual cells from transgenic mice harboring a fluorescent protein reporter.
Genesis
27:
95-98,
2000[ISI][Medline].
16.
Haugland, RP.
Handbook of Fluorescent Probes and Research Chemicals. Eugene, OR: Molecular Probes, 2002, sect. 20.4.
17.
Jensen, J,
Heller RS,
Funder-Nielsen T,
Pedersen EE,
Lindsell C,
Weinmaster G,
Madsen OD,
and
Serup P.
Independent development of pancreatic - and
-cells from neurogenin3-expressing precursors. A role for the notch pathway in repression of premature differentiation.
Diabetes
49:
163-176,
2000[Abstract].
18.
Jiang, Y,
Jahagirdar BN,
Reinhardt RL,
Schwartz RE,
Keene CD,
Ortiz-Gonzalez XR,
Reyes M,
Lenvik T,
Lund T,
Blackstad M,
Du J,
Aldrich S,
Lisberg A,
Low WC,
Largaespada DA,
and
Verfaillie CM.
Pluripotency of mesenchymal stem cells derived from adult marrow.
Nature
418:
41-49,
2002[ISI][Medline].
19.
Kanno, T,
Göpel SV,
Rorsman P,
and
Wakui M.
Cellular function in multicellular system for hormone-secretion: electrophysiological aspect of studies on -,
- and
-cells of the pancreatic islet.
Neurosci Res
42:
79-90,
2002[ISI][Medline].
20.
Kurebayashi, N,
Harkins AB,
and
Baylor SM.
Use of fura red as an intracellular calcium indicator in frog skeletal muscle fibers.
Biophys J
64:
1934-1960,
1993[Abstract].
21.
Lacy, PE,
and
Kostianovsky M.
Method for the isolation of intact islets of Langerhans from the rat pancreas.
Diabetes
16:
35-39,
1967[ISI][Medline].
22.
Lumelsky, N,
Blondel O,
Laeng P,
Velasco I,
Ravin R,
and
McKay R.
Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets.
Science
292:
1389-1394,
2001
23.
Meyer, K,
Irminger J-C,
Moss LG,
de Vargas LM,
Oberholzer J,
Bosco D,
Morel P,
and
Halban PA.
Sorting human -cells consequent to targeted expression of green fluorescent protein.
Diabetes
47:
1974-1977,
1998[Abstract].
24.
Morgan, CR,
and
Lazarow A.
Immunoassay of insulin: two antibody system. Plasma insulin levels in normal, subdiabetic, and diabetic rats.
Diabetes
12:
115-126,
1963[ISI].
25.
Palmiter, RD,
Sandgren EP,
Avarbock MR,
Allen DD,
and
Brinster RL.
Heterologous introns can enhance expression of transgenes in mice.
Proc Natl Acad Sci USA
88:
478-482,
1991[Abstract].
26.
Postic, C,
Shiota M,
Niswender KD,
Jetton TL,
Chen Y,
Moates JM,
Shelton KD,
Lindner J,
Cherrington AD,
and
Magnuson MA.
Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic cell-specific gene knock-outs using cre recombinase.
J Biol Chem
274:
305-315,
1999
27.
Scearce, LM,
Brestelli JE,
McWeeney SK,
Lee CS,
Mazzarelli J,
Pinney DF,
Pizarro A,
Stoeckert CJ, Jr,
Clifton SW,
Permutt MA,
Brown J,
Melton DA,
and
Kaestner KH.
Functional genomics of the endocrine pancreas: the pancreas clone set and PancChip, new resources for diabetes research.
Diabetes
51:
1997-2004,
2002
28.
Tsien, RY.
The green fluorescent protein.
Annu Rev Biochem
67:
509-544,
1998[ISI][Medline].
29.
Van de Winkel, M,
Maes E,
and
Pipeleers D.
Islet cell analysis and purification by light scatter and autofluorescence.
Biochem Biophys Res Commun
107:
525-532,
1982[ISI][Medline].
30.
Wu, Y,
and
Clusin WT.
Calcium transient alternant in blood-perfused ischemic hearts: observations with fluorescent indicator fura red.
Am J Physiol Heart Circ Physiol
273:
H2161-H2169,
1997