Detection of insulin-releasing cells using in situ immunoblotting

www.elsevier.com/locate/yabio

ANALYTICAL

BIOCHEMISTRY

Analytical Biochemistry 366 (2007) 137–143

Detection of insulin-releasing cells using in situ immunoblotting

Hao Chen, Hideki Sato, Takahiko Totani, Hiroo Iwata *

Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawara-Cho, Shogoin, Sakyo-Ku, Kyoto 606-8507, Japan

Received 21 November 2006Available online 12 April 2007

Abstract

Embryonic stem (ES) cells hold promise as a source for cell transplantation treatment of diseases such as type I diabetes. Further, cellsreleasing bioactive substances from ES cell progeny may be concentrated and purified for clinical applications. Although ES cell linesthat express reporter genes have been established to isolate cells releasing bioactive substances, other difficulties must be overcome beforethese genetically modified cells can be used for gene therapy in human patients. Fluorescence- or magnetic-activated cell sorters are com-monly used to isolate specific cells using antibodies against cell surface antigens. However, for some cells, such as insulin-producing betacells, specific surface antigens have not yet been identified. In this study, we developed a simple and efficient method to identify and purifyinsulin- and alpha-fetoprotein-producing cells. A nitrocellulose membrane treated with anti-insulin or anti-alpha–fetoprotein antibodieswas placed on a cell layer to trap insulin or alpha-fetoprotein released from the cells. The location of specific substance-producing cellswas identified by immunostaining the membrane. The insulin-releasing cells were selectively collected from the culture dish using a clon-ing ring and transferred to another culture plate.� 2007 Elsevier Inc. All rights reserved.

Keywords: Immunoblotting; Insulin; Cell cloning; Stem cells

Many diseases are caused by the disrupted function ofparticular cells in the human body. For instance, type I dia-betes is caused by the destruction of pancreatic beta cells,and Parkinson’s disease is caused by the degeneration ofdopaminergic neurons. Transplantation of functional insu-lin- or dopamine-releasing cells isolated from human tissuehas been performed in clinics to treat patients [1,2]. How-ever, due to the shortage of human organ or tissue donors,the number of patients treated with cell transplantation hasbeen limited. New cell sources, such as embryonic stem(ES)1 cells or somatic stem cells, are currently beinginvestigated.

ES cells can proliferate indefinitely and can differentiateinto the three embryonic germ layers: endoderm, meso-

0003-2697/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2007.04.008

* Corresponding author. Fax: +81 75 751 4646.E-mail address: [email protected] (H. Iwata).

1 Abbreviations used: ES, embryonic stem; FACS, fluorescence-activatedcell sorters; MACS, magnetic-activated cell sortess; AFP, alpha-fetopro-tein; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline; HRP, horseradish peroxidase.

derm, and ectoderm [3]. Because they are pluripotent, EScells have been thought to be a promising cell source forcellular therapeutic applications. Many studies with mouseand human ES cells have demonstrated effective derivationof insulin-releasing cells [4–6]. In our previous work, wederived ES cell progeny, including many colonies of insu-lin- and C-peptide-positive cells, from mouse ES cells.The progeny still contained insulin- and C-peptide-positivecells after nine rounds of subculturing, but the populationof insulin-releasing cells did not increase [7]. It has beenreported that beta cells are capable of self-renewal [8–10].If progenitor or immature insulin-releasing cells with pro-liferation potential could be isolated from ES cell progeny,a large number of insulin-producing cells could be obtainedby in vitro cell expansion. Several methods to isolate spe-cific cells have been developed. ES cell lines that carry areporter gene have been established to isolate insulin-pro-ducing cells [11,12], but other difficulties must be overcomebefore these genetically modified cells can be used as genetherapy in human patients. Fluorescence- or magnetic-acti-vated cell sorters (FACS or MACS) are commonly used to

mailto:[email protected]

138 In situ immunoblotting / H. Chen et al. / Anal. Biochem. 366 (2007) 137–143

isolate specific cells using antibodies against cell surfaceantigens. However, surface antigens specific to beta cellsthat can be used in FACS and MACS have not yet beenidentified. We wished to develop a simple and efficientmethod to identify and purify insulin-producing cells.

In this study, we developed a simple method based onin situ immunoblotting to identify and isolate bioactivesubstance-producing cells from a tissue culture dish. Its effi-cacy was demonstrated using insulin-producing MIN6 cellsand alpha-fetoprotein (AFP)-producing Hep-G2 cells asmodel cell cultures. The cell spots were placed in an orderlyarray on the culture dishes. A nitrocellulose membranecoated with anti-insulin or anti-AFP antibodies was gentlyplaced on the cell layer to bind to insulin or AFP releasedfrom the cells. The location of insulin- or AFP-producingcells was identified by immunostaining the membrane todetect the bound insulin or AFP. The insulin-producingcells could be selectively collected from the culture dishusing the cloning ring method and replated onto anotherculture dish.

Materials and methods

Cell culture

Immortalized mouse pancreatic beta cells, MIN6 [13],were kindly donated by Dr. Miyazaki (Osaka University,Osaka, Japan). Immortalized human hepatoma Hep-G2cells, which secrete AFP, were obtained from the HealthScience Research Resources Bank (Osaka, Japan). Bothof these cell lines were routinely maintained using DMEM(Invitrogen Corp., Carlsbad, CA) supplemented with 10%fetal bovine serum and antibiotics (10 U/ml penicillin,and 10 mg/ml streptomycin) in a humidified atmosphereof 5% CO2/95% air at 37 �C.

Preparation of the silicon sheet

A 76-mm · 26-mm slide glass (Matsunami Glass Ind.,Ltd. Osaka, Japan) was cut in half (see Scheme 1). It waswashed with ethanol (Nacalai Tesque, Inc., Kyoto, Japan)and distilled water, dried by N2 blowing, and then coatedwith Barrier Coat (Shin-Etsu Chemical Co., Tokyo, Japan)using a spincoater for 10 s at 1000 rpm followed by incuba-tion at 120 �C for 3 min. A frame made of a 1.0-mm-thicksilicon sheet (As One Corp., Osaka, Japan) was put on theslide. A 10:1 mixture of a dimethylsiloxane prepolymer anda curing agent (Sylgard 184 Silicone Elastomer Kit, TorayIndustries, Inc., Tokyo, Japan) was poured into the frameon the glass slide. The slide was left in a desiccator underreduced pressure for 45 min to remove air from the mix-ture. After another glass slide coated with Barrier Coatwas placed on the mixture, it was cured at 120 �C for 45min. The cured silicon sheet was removed from the glassplate and nine holes (1.9 mm in diameter) were punchedout of the silicon sheet using a stainless steel pipe(1.9 mm inside diameter).

Spotting cells on a culture dish

The silicon sheet with nine holes was rinsed with 70%ethanol and placed in a 6-cm culture dish. Five microlitersof a cell suspension containing 9000 MIN6 or Hep-G2 cellswas put into each hole of the silicon sheet. After 700 ll ofphosphate-buffered saline (PBS) was left on the peripheryof the dish to suppress evaporation of the culture medium,the dish was incubated for 6–7 h at 37 �C to allow cells tosettle on the surface of the dish. PBS was removed from thedish and 7 ml culture medium was added on top of the sil-icon sheet. The dish was left overnight in a humidifiedatmosphere of 5% CO2/95% air at 37 �C. After cell adhe-sion onto the dish was confirmed, tweezers were used toremove the silicon sheet from the dish, using care not todisturb the cell spots.

To examine the sensitivity, 5 ll of a cell suspension con-taining 1000, 3000, 6000, or 9000 MIN6 cells was put intoeach hole of the silicon sheet and the dish was incubated for7 h at 37 �C to allow cells to settle on the surface of the dishas described above. After the silicon sheet was removed,4 ml of a cell suspension containing 1 · 106 Hep-G2 cellswas applied into the culture dish.

In situ immunoblotting was performed after 11 h addi-tional culture as follows.

In situ immunoblotting

The outline of the immunoblotting procedure is shownin Scheme 1. A nitrocellulose membrane coated with anti-bodies against human insulin or against human AFP wasprepared as follows. A plain nitrocellulose membrane(Bio-Rad Laboratories, Inc., CA) was treated with mouseanti-human insulin monoclonal antibody in PBS (diluted1:200, Sigma–Aldrich Co., St. Louis, MO) or mouse anti-human AFP monoclonal antibody in PBS (diluted 1:200,Acris Antibodies GmbH., Himmelreich, Germany) for1 h at 37 �C. The membrane was blocked with a solutionof 3% nonfat milk in (10 mM Tris–HCl, 100 mM NaCl,0.1% Tween, pH 7.5), (TTBS) for 1 h at 37 �C and thenwashed twice with PBS.

For in situ immunoblotting, the membrane carryinganti-insulin antibody or anti-AFP antibody should beplaced gently on top of the cell layer. When a membranedirectly touches the cell layer, the cells attach to the mem-brane and will be removed from the culture dish. In Teruyaet al.’s study [14], a filter paper was inserted between themembrane and the cells; however, this procedure causedcells to deteriorate. Therefore, instead of using a filter toform a layer between the membrane and the cells, in thisstudy we used a highly viscous culture medium preparedby adding 2% methylcellulose to the medium. The culturemedium was removed from a dish that had been placedover the cells, and 1 ml of DMEM supplemented with 2%methylcellulose was added to the culture. The thicknessof the medium layer was estimated to be about 470 mm,calculated using the volume of the culture medium added

1.9 mm

5.0 mm

Guinea pig anti-insulin polyclonal antibody

Donkey anti-guinea pig IgG HRP conjugated monoclonal antibody

Reversed

Scheme 1. In situ immunoblotting procedure for identifying cells releasing insulin or alpha–fetoprotein.

In situ immunoblotting / H. Chen et al. / Anal. Biochem. 366 (2007) 137–143 139

(1 ml) and the surface area of the culture dish (21.3 cm2).The nitrocellulose membrane carrying anti-human insulinantibody or anti-human AFP antibody was layered overthe medium. After a 1-h incubation at 37 �C, 5 ml of cul-ture medium without methylcellulose was infused under-neath the membrane, and the membrane was carefullyrecovered with tweezers and washed twice with TTBS.

Immunostaining was used to determine the position ofimmobilized proteins on the membrane. The membranewas treated with guinea pig anti-swine insulin polyclonalantibody (diluted 1:100; Dako Japan Inc., Kyoto, Japan)or with rabbit anti-human AFP polyclonal antibody(diluted 1:100; Sanbio BV, Uden, Netherlands) dilutedwith Can Get Signal Solution1 (Toyobo Co., Ltd., Osaka,Japan) for 1 h at room temperature. After washing withTTBS, the membrane was incubated with donkey anti-gui-nea pig IgG(H+L) antibody conjugated with horseradishperoxidase (HRP) (diluted 1:5000; Chemicon Int. Inc.,Temecula, CA) or goat anti-rabbit IgG(H+L) antibodyconjugated with HRP (diluted 1:2500; Jackson ImmunoRe-search Laboratories, Inc., West Grove, PA) in Can GetSignal Solution2 for 1 h at room temperature. Finally,color was developed using Immunostain HRP-1000(Seikagaku Corp., Tokyo, Japan).

Immunochemical staining of cells on culture dishes

Cultured cells were fixed with 4% paraformaldehydesolution for 15 min at room temperature in culture dishes,permeabilized with 0.2% Triton X-100 in PBS, and thenblocked with 2% nonfat milk in PBS for 1 h at room tem-perature. The cells were treated with a mixture of guineapig anti-swine insulin polyclonal antibody (diluted 1:100)and rabbit anti-human AFP polyclonal antibody (diluted1:200) in 2% nonfat milk in PBS at 4 �C overnight. Afterwashing with 0.05% Tween-20 in PBS (TPBS), the cells

were treated with a mixture of Alexa488-conjugated goatanti-guinea pig IgG(H+L) antibody (diluted 1:500, Invitro-gen Corp.) and Alexa594-conjugated goat anti-rabbitIgG(H+L) antibody (diluted 1:500, Invitrogen Corp.) in2% nonfat milk in PBS for 2 h. After washing with TPBS,the cells were treated with Hoechst 33342 fluorescent dye(Dojindo Laboratories, Kumamoto, Japan) for nuclearDNA staining.

Collection of insulin-positive cells

Images of the immunostained membranes were collectedwith a scanner (ES-8000; Seiko Epson Corp., Nagano,Japan). The captured image was reversed by using AdobePhotoshop version 5.0 (Adobe Systems Inc., San Jose,CA). The reversed image was printed and placed underthe culture dish. The cells at the immunopositive positionswere collected as follows. The culture medium wasremoved. Then cloning ring (inside diameter 3.4 mm, AsahiTechnoglass Corp., Chiba, Japan) with silicon grease(Shin-Etsu Chemical Co.) at its rim was put on the dishat the immunopositive spot. A 0.25% trypsin EDTA solu-tion (Nacalai Tesque) was added into the cloning rings todetach cells from the culture dish. Trypsin activity washalted by adding culture medium containing serum intothe cloning rings and the cells were collected. The cells wereseeded into a well of a 96-well multiwell plate. These col-lected, plated cells were analyzed by immunostaining usingthe method described above for cells on a culture dish.

Results and discussion

Mouse MIN6 cells, which produce insulin, and humanHep-G2 cells, which produce AFP, were used as modelcells to develop an in situ immunoblotting method fordetecting and isolating protein-secreting cells from cell

Fig. 1. Cell morphology before and after in situ immunoblotting. (A) and (B) MIN6 cells; (C) and (D) Hep-G2 cells. Scale bars, 200 lm.

Fig. 2. Nitrocellulose membranes used for immunoblotting insulin or alpha–fetoprotein, immunohistochemical staining of cell spots on a culture dish, andcollection of MIN6 cells from a spot identified using the in situ immunoblotting method. (A) Membrane used for immunoblotting insulin. (B) Membraneused for immunoblotting alpha–fetoprotein. (C) Cell spots stained with anti-insulin (green) and anti-AFP (red) polyclonal antibodies. Scale bars in A, B,and C, 1 cm. MIN6 cells were collected from the spot using trypsin and a cloning ring and were replated in a well of a 96-well plate. After incubation for 2days, cells were immunostained using anti-insulin antibody. (D) Immunostaining image for insulin (green). (E) Nuclear staining image with Hoechst 33342(blue). (F) Merged image from D and E. Scale bars in D, E, and F, 100 lm.



culture plates. The cells were spotted on a culture dish. Inthis experiment, five of the nine cell spots were MIN6 cells,and the other four spots were Hep-G2 cells. Highly viscousculture medium, prepared by adding 2% methylcellulose tomedium, was added to the culture dish to form a thin layerof medium over the cell spots. A nitrocellulose membrane,previously coated with antibodies that recognize insulin orAFP, was gently placed on top of the cell spots but did notdirectly contact the cells. The viscous medium served as a470-lm cushion between the cells and the membrane, pre-venting the cells from binding to the membrane. After a 1 hincubation at 37 �C, the membrane was carefully removedand immunostained for insulin or AFP. After removal ofthe membrane, the culture dish was washed with culturemedium without methylcellulose and observed by phasecontrast microscopy. As shown in Fig. 1, no cell damage

Fig. 3. Nitrocellulose membranes used for immunoblotting insulin and immunfor immunoblotting insulin. Scale bar, 2 mm. (B) Merged image with the imagwith Hoechst 33342 (blue). Scale bars, 500 lm. P1–P6 represent positions 1–6.1000 cells respectively.

was observed for either MIN6 cells or Hep-G2 cells aftera series of incubations with membranes.

Insulin and AFP, which were secreted from the cells anddiffused to the nitrocellulose blotting membrane, bound tomembranes carrying anti-insulin or anti-AFP antibodies,respectively. Those spots were visualized by immunostain-ing, as shown in Fig. 2A and B. To verify the correlation ofimmunoblotted spots on the membrane and cell spots onthe culture dish, the cells on the culture dish were fixedand immunostained using anti-insulin antibody and anti-AFP antibody after removal of the membrane. Fig. 2Cshows photos of the immunostained culture dish. Insulin-positive (green) and AFP-positive (red) spots can clearlybe seen on the culture dish. The immunoblots on the mem-brane shown in Fig. 2A and B formed mirror images of thecell spots on the culture dish (Fig. 2C).

ohistochemical staining of cell spots on a culture dish. (A) Membrane usede of immunostained with anti-insulin (green) and that of nuclear stainingSpots P1, P2, P3, P4, P5, and P6 contained 1000, 6000, 0, 3000, 9000, and


The aim of this study was to develop a method to isolatebioactive substance-producing cells from the progeny ofstem cells. We have already described the method (in situ

immunoblotting) used to locate the specific cells. Themethod used to retrieve the cells from a culture dish isshown schematically in Scheme 1. The reversed image ofthe immunoblotted membrane was printed and placedunder the culture dish. A cloning ring was placed on cellslocated on the insulin-positive blots, and trypsin solutionwas added to the ring. Cells were collected from the dish.The viability of the collected MIN6 cells was greater than90%, as determined by the trypan blue exclusion test. Thecollected MIN6 cells were reseeded in a well of a 96-wellplate and cultured for 2 days in DMEM in 5% CO2 at37 �C. Micrographs of immunohistochemical staining andnuclear staining of the MIN6 cells are shown in Fig. 2F.MIN6 cells adhered well to the dish and formed cell clus-ters. This indicated that insulin releasing cells could befound and isolated by the combination of the in situ immu-noblotting and the cloning ring procedure.

The limit of detection of the method and the effect ofcoexistence of other kinds of cells on the in situ immuno-blotting were examined. Different numbers of MIN6 cells(1000, 3000, 6000 and 9000 cells) were put into each holeof the silicon sheet and left for 7 h to adhere to the culturedish. After the silicon sheet was removed, Hep-G2 cellswere applied into the culture dish. In situ immunoblottingwas performed after 11 h additional culture. The five posi-tive spots could be seen on the membrane as shown inFig. 3A. Fig. 3 also includes micrographs of immunohisto-chemical insulin staining and nuclear staining of the spots.The coexistence of Hep-G2 cells in the spots did not inter-fere with the in situ immunoblotting. Color density of each

Scheme 2. Secreted substance released from a cell diffuses to the membrane. (Acloud with radius Rt. (B) The substance is released from a cell spot with radiusRt. The diameter of the immunoblotted spot is expressed as diameter = 2R =

spot on the immunoblotting membrane was correlated withthe number of MIN6 cells in each spot. The color densityincreased with increasing numbers of MIN6 cells in thespot. As seen in Fig. 3A, 3000 MIN6 cells in a spot(1.9 mm in diameter) could be easily detected. The spotcontaining 1000 MIN6 cells might be possibly found. Thus1000 MIN6 cells in a spot (1.9 mm in diameter) are takenas the limit of detection by this method.

In our experimental setup, insulin and AFP releasedfrom the cells traveled by diffusion to the blotting mem-brane and were trapped by the antibodies on the mem-brane. The time needed for one-dimensional diffusionover distance L can be estimated using the following equa-tion, in which t represents time (in seconds) and D repre-sents the diffusion coefficient (cm2/s):

t ¼ L2=2D: ð1Þ

The cells and the membrane were separated by 470 lm ofculture medium, and the diffusion coefficients of insulinand AFP in water at 20 �C are D = 7.3 · 10�7 cm2/s andD = 6.6 · 10�7 cm2/s [15], respectively. Thus, the time re-quired for diffusing 470 lm is estimated using Eq. (1) tobe about 30 min for both insulin and AFP. The blottingmembrane was left on the culture dish for 1 h allowing suf-ficient time for the diffused proteins to bind to the blottingmembrane.

Note that the diameters of the spots on the immunoblots(3 mm in diameter) are about 1.6 times larger than those ofthe cell spots on the culture dish. When insulin and AFPdiffuse vertically to the membrane, they also diffuse later-ally. Suppose that a single cell adheres to a culture dishand continuously releases a substance as shown in Scheme2A. Diameter of the area containing the diffused substance

) The substance is released from the cell. After t s, the substance forms aR0. A cell at the periphery of the cell culture spot forms a cloud with radius2R0 + 2Rt.


after time t is approximately expressed by Eq. (2) for two-dimensional diffusion, in which R is the radius in cm (anddiameter is 2 · R):

t ¼ R2t =4D ð2Þ

In our experimental setup, each spot contained more than9000 cells (1.9 mm in diameter). Referring to Scheme 2, asubstance released by cells at the periphery of the cell spotdiffuses a distance of Rt during time, t. Thus, the diameterof the immunoblotted spots can be roughly expressed by

2R ¼ 2R0 þ 2Rt; ð3Þwhere R0 is the radius of the cell spot, Rt is obtained fromEq. (2), and R is the radius of a blotted spot on the mem-brane at time t. In this experiment, the diameter of theimmunopositive spot on the immunoblot, 2R, is estimatedto be about 3.7 mm after 1 h blotting using Eqs. (2) and (3)and diffusion coefficient. This theoretical value is in agree-ment with the experimental diameter of the spots observedon the immunoblotted membrane. To obtain a blottingspot similar in size to that of the cell spot, the distance be-tween the cell layer and the blotting membrane should beas small as possible.

Conclusion

In this study, we developed a simple and efficientmethod to identify and purify insulin-producing cells. Anitrocellulose membrane coated with anti-insulin antibodywas placed gently on a cell layer to trap insulin releasedfrom the cells. The location of the cells releasing the insulinwas determined by immunostaining the membrane-boundinsulin. The insulin-releasing cells could then be selectivelycollected from the dish by trypsin treatment using a cloningring. The method developed in this study may be useful forpurifying and concentrating insulin-releasing cells fromstem cells progeny for clinical applications.

References

[1] A.M. Shapiro, J.R. Lakey, E.A. Ryan, G.S. Korbutt, E. Toth, G.L.Warnock, N.M. Kneteman, R.V. Rajotte, Islet transplantation in

seven patients with type 1 diabetes mellitus using a glucocorticoid-freeimmunosuppressive regimen, N. Engl. J. Med. 343 (2000) 230–238.

[2] C.R. Freed, P.E. Greene, R.E. Breeze, W.Y. Tsai, W. DuMouchel, R.Kao, S. Dillon, H. Winfield, S. Culver, J.Q. Trojanowski, D.Eidelberg, S. Fahn, Transplantation of embryonic dopamine neuronsfor severe Parkinson’s disease, N. Engl. J. Med. 344 (2001) 710–719.

[3] J.A. Thomson, J. Itskovitz-Eldor, S.S. Shapiro, M.A. Waknitz, J.J.Swiergiel, V.S. Marshall, J.M. Jones, Embryonic stem cell linesderived from human blastocysts, Science 282 (1998) 1145–1147.

[4] S. Assady, G. Maor, M. Amit, J. Itskovitz-Eldor, K.L. Skorecki, M.Tzukerman, Insulin production by human embryonic stem cells,Diabetes 50 (2001) 1691–1697.

[5] N. Lumelsky, O. Blondel, P. Laeng, I. Velasco, R. Ravin, R. McKay,Differentiation of embryonic stem cells to insulin-secreting structuressimilar to pancreatic islets, Science 292 (2001) 1389–1394.

[6] Y. Moritoh, E. Yamato, Y. Yasui, S. Miyazaki, J. Miyazaki, Analysisof insulin-producing cells during in vitro differentiation from feeder-free embryonic stem cells, Diabetes 52 (2003) 1163–1168.

[7] T. Ibii, H. Shimada, S. Miura, E. Fukuma, H. Sato, H. Iwata,Possibility of insulin-producing cells derived from mouse embryonicstem cells for diabetes treatment, J. Biosci Bioeng. 103 (2007) 140–146.

[8] S. Bonner-Weir, Beta-cell turnover: its assessment and implications,Diabetes 50 (Suppl. 1) (2001) S20–S24.

[9] G.M. Beattie, A.M. Montgomery, A.D. Lopez, E. Hao, B. Perez,M.L. Just, J.R. Lakey, M.E. Hart, A. Hayek, A novel approach toincrease human islet cell mass while preserving beta-cell function,Diabetes 51 (2002) 3435–3439.

[10] Y. Dor, J. Brown, O.I. Martinez, D.A. Melton, Adult pancreaticbeta-cells are formed by self-duplication rather than stem-celldifferentiation, Nature 429 (2004) 41–46.

[11] B. Soria, E. Roche, G. Berna, T. Leon-Quinto, J.A. Reig, F. Martin,Insulin-secreting cells derived from embryonic stem cells normalizeglycemia in streptozotocin-induced diabetic mice, Diabetes 49 (2000)157–162.

[12] Y. Moritoh, E. Yamato, Y. Yasui, S. Miyazaki, J. Miyazaki, Analysisof insulin-producing cells during in vitro differentiation from feeder-free embryonic stem cells, Diabetes 52 (2003) 1163–1168.

[13] J. Miyazaki, K. Araki, E. Yamato, H. Ikegami, T. Asano, Y.Shibasaki, Y. Oka, K. Yamamura, Establishment of a pancreatic betacell line that retains glucose-inducible insulin secretion: specialreference to expression of glucose transporter isoforms, Endocrinol-ogy 127 (1990) 126–132.

[14] K. Teruya, S. Shirahata, T. Yano, K. Seki, H. Tachibana, H. Ohashi,H. Murakami, Secretory cell immunoscreening assay–a highly sensi-tive screening method for secretory cells, Anal. Biochem. 214 (1993)468–473.

[15] S. Aliau, J. Marti, J. Moretti, Bovine alpha-fetoprotein. Isolation andcharacterization, Biochimie 60 (1978) 663–672.

Detection of insulin-releasing cells using in situ immunoblotting

Documents

Transcript of Detection of insulin-releasing cells using in situ immunoblotting