Islets of Langerhans - Science topic

1. WORLD HEALTH ORGANIZATION. Diabetes – Fact Sheet, 2013. Disponível em: <http://www.who.int/mediacentre/factsheets/fs312/en/index.html> 2. WORLD HEALTH ORGANIZATION. Noncommunicable diseasescountry profile – Brazil. 2010. Disponível em: <http://www.who.int/nmh/countries/bra_en.pdf> 3. SOCIEDADE BRASILEIRA DE DIABETES. Diretrizes da Sociedade Brasileira de Diabetes, 2009. Disponível em: <http://www.diabetes.org.br/attachments/diretrizes09_final.pdf> 4. EUROPEAN MEDICINES AGENCY. European Medicines Agency 2011 Priorities for Drug Safety Research - Anti diabetic drugs: Cardio/cerebrovascular adverse effect and pancreatitis/ pancreatic cancer. Julho/2010. Disponível em: <http://www.ema.europa.eu/docs/en_GB/document_library/Other/2010/07/WC500094264.pdf> 5. BUTLER, A. E. et al. Marked Expansion of Exocrine and Endocrine Pancreas with Incretin Therapy in Humans with increased Exocrine Pancreas Dysplasia and the potential for Glucagon-producing Neuroendocrine Tumors. 22 Março 2013. Disponível em: <http://diabetes.diabetesjournals.org/content/early/2013/03/17/db12-1686.abstract> 6. U.S. FOOD AND DRUG ADMINISTRATION. Incretin Mimetic Drugs for Type 2 Diabetes: Early Communication - Reports of Possible Increased Risk of Pancreatitis and Precancerous Findings of the Pancreas. Março/2013. Disponível em: <http://www.fda.gov/safety/medwatch/safetyinformation/safetyalertsforhumanmedicalproducts/ucm343805.htm> 7. EUROPEAN MEDICINES AGENCY. Assessment report for GLP-1 based therapies. 25 Julho 2013. Disponível em: <http://www.ema.europa.eu/docs/en_GB/document_library/Report/2013/08/WC500147026.pdf> 8. AGÊNCIA NACIONAL DE VIGILÂNCIA SANITÁRIA. Anvisa esclarece questões sobre indicação e segurança do medicamento Victoza (Liraglutida) - Informe SNVS/Anvisa/Nuvig/GFARM nº 07, de 06 de setembro de 2011. Disponível em: <http://s.anvisa.gov.br/wps/s/r/bay7>

I am looking for a surface membrane protein (which is not present on iPSCs, pancreatic progenitors or any mature islet cells) which I could express as a selectable marker. My cells have a tendency to differentiate in Puromycin and other antibiotics and I am already using fluorophores as selectable markers for doxycyclin-induced cells, so having a unique protein expressed on the cell surface that I can pick up with an antibody and flow cytometry would be very helpful.

After cross referencing alginate hydrogel properties (common material used in islet cells encapsulation), I saw some of its property have similarities with honey. I wonder are there studies or research about the effect of honey on islet cells if you will observe them by putting islet cells on a petri dish with honey?

Currently, I'm working on my senior research project that involves IHC staining of islets of Langerhans in rats. Prior to implantation (see BVP 8 CTL image attached), most of the islets demonstrate insulin staining (red) and good nuclear staining via DAPI (blue). However, in the explants after about 2-3 months of implantation (see BVP13/14 Explant image attached), the insulin stain remains in what we assume to be islets, but the nuclear staining is non-existent in these "islets". Has anyone experienced a similar problem in nuclear staining in explants? Or in islets? The green stain is for CD31, an endothelial marker, if that information helps.

Thank you for any help!

tags: islets of Langerhans, pancreas, implantation, explants, DAPI, staining, nuclear staining, lack of DAPI, insulin, CD31

I am trying to see the expression of Bim - BH3 only pro-apoptotic protein in dispersed mouse islet cells (seeded at 30,000 cells/well or 40,000 cells/well).

I have tried qPCR and western blot to check for the expression of Bim, but I haven't got any results. I am just wondering what is going wrong?

Thank you for your help in advance.

I am doing my research on islet cells. i want to check the insulin secretion in vitro. are there diabetic induced pancreatic beta cell lines available? if so what is the best?

I would like to do an experiment on the beta cells in which I would like to know what is the external pH surrounding the cells

Gene therapy to regenerate islets!!! Congratulations!!

I read that you culture cells on a non-adherent surface to promote pseudoislet formation, but won't the pseudoislets break apart after you spin them down and resuspend the cells?  What type of ratio do you subculture at and how confluent do you plate the cells at?

With respect to beta cells, do MIN6 form pseudoislets best?  I've seen INS-1E in publications, but do parental INS1 and clonal 832/13 also form pseudoislets well?

I plan to do pSTAT-3 (Tyr705) (and also pGSK3B and later pSTAT1) western-blot experiments on rat islets and INS1 pancreatic beta cells. To activate the STAT3 signaling pathway, I will use a combination of IL1B, TNFa and INFg (I’m not sure that it will really promote STAT3 phosphorylation),and I’ll use pSTAT3 antibody (ref. 9131) from Cell Signaling.

As I know it’s necessary to be very carefull to preserve protein phosphorylation state, I’d like to have your advice to do this kind of experiment, for instance on the following points :

- serum (and even Glucose ?) deprivation steps, or even KREBS pre-incubation steps ? during how much time?

- very soft centrifugation or even no centrifugation (for islets) (but which would imply not doing washing steps before lysis?)

- special lysis buffer (I plan to use Sigma Proteases (PIC) and Phophatases (PIC2 and PIC3) Inhibitors Cocktails) ?

- avoiding sonication steps (which are usually necessary to extract well protein from islets)?

- minimum amount of protein per well for the western-blot ?

- using BSA instead of milk for primary antibody incubation (as mentioned on the data sheet ) but also for saturation?

Thanks you very much by advance for your help !

I have a mouse model that expresses modified Cre recombinase, so that only upon tamoxifen treatment of the animals they express Cre, thus KO of floxed gene can be induced. I want to isolate the islets of Langerhans from this mouse model and treat them in vitro with 4-hydroxy tamoxifen (4-HT). If anybody can give me suggestions as to how to treat islets in culture with 4- HT? 

If this works what kind of tranfection reagent is recommended for the islets?

Does anybody know a good UCP2 Antibody for murine Islets of Langerhans?

Has anyone handled Liberase TL or Collagenase? I dissolve and aliquote Liberase with water. Then I free free and store the enzyme in liquid nitrogen. The question is how to thaw them to keep them active? One paper said they thaw it on ice, while other said it should be under hot water to get quick thawing. Which one is better? Liberase is similar as collagenase. Any experience using collagenase should also be useful.

It is generally agreed that insulin secretion increases as blood glucose increases, and the same is true for islets/beta-cells in a dish in response to glucose. I have been trying to locate definitive findings in the literature of what the glucose dose-response curve for insulin secretion looks like for rodent and/or human islets across a broad range of glucose concentrations (0-200 mM or more). At what glucose load does insulin secretion max out? My impression is that the range is far broader than I would have expected. Does secretion remain steady beyond this peak value or does it actually diminish if glucose loads become too high, thus creating the inverted U-shaped curve? I would greatly appreciate citations or links to well accepted publications on this matter.

i was looking for the easiest method to count islet langerhans of a mice which is diabetes induced by stz. I have found so many paper which work on that topics, but i found that the quantitative method in scoring histological islet is difficult to do in my lab, since we don't have any imaging program. so i would like to ask about the simplest way to do semikuantitative/kuantitative count on pancreatic islet mice, any one can help? i've found paper which doing semikuantitave scoring, but it is for fibrosis/lesion mice, i am afraid that definition is not appropriate for the mechanism of stz in doing destruction to mice pancreas

I wanted to attach pancreatic islets to the bottom of the culture dish, " How can i achieve this? Normally i am using matrigel for cells but the method is not working for islets... Please give some suggestions

I am working on a project related to pancreatic beta cells. I would like to know what is the specific impact of diabetes on electrical patterns of beta cell activities. I know the diabetes reduces the amplitude of bursting and bursting mass but what about the burst duration, inter-BRST interval and the ability of disease on changing bursting status to spiking behavior?

What is the defect of pancreatic beta cells if the glucose stimulated insulin secretion process is not functional, but it still responds to Ionomycin, and has no response to 30mM KCl induced depolarization.

Has anyone ever handled Liberase TL? I dissolved enzyme powder and aliquot. After that, I quick-froze and stored them in liquid nitrogen. My question is how do I thaw it to keep its activity? I saw a protocol which said thaw the enzyme on ice. But some others said that the enzyme should be thawed under hot water just as thawing a cell line. So which method is better? Thanks!

We are preparing gradient solutions of Ficoll dissolved in Eurocollins with some steps of stirring at 50-70°C and also autoclaving.

A few months ago, I submitted my paper to a journal. One of my manuscript's reviewers asked me to quantify the HE and IHC staining of the pancreatic tissue. Unfortunately, now I have no software to quantify that data. So, I humbly request you to suggest me some online tools to quantify that data? Thank you!

I read the articles regarding preparation KRBB for isolated islets experiment, but I don't know the principle behind this procedure. Could anyone can let me know why we need to prepare the gas 95%O2/5%co2 for KRBB? what if not? Thanks

We are thinking about doing a rescue gradient purification of human islets by discontinuous gradients and we would like to prepare them with Ficoll powder in Eurocollins.

I performed an Insulin Sensitivity Test on diabetic and control mice. I noticed that after 30 minutes blood glucose level come back to the levels before the insulin administration and this is happening faster in our diabetic mice. This can't only be explained by the turnover of insulin. Does somebody have an idea? Can it be an uncontrolled activity of glycogenolysis and gluconeogenesis?

Has anyone tried BSA gradient for islet separation? Please share details.

I would like to prepare solutions of: 1.108,1.096 and 1.037 g/ml from a stock of Ficoll 1.132. Thanks.

I need to freeze isolated islets from human pancreas. The islets grown in CMRL 1066 medium supplemented with added vitamin E, nicotinamide, heparin, albumin, and ciprofloxacin. Some authors use RPMI 1640 with FCS and 10% DMSO, others use Medium 199 with added FCS and 10% DMSO. With my islets growing in CMRL 1066, I would like to know if I can use the same medium for freezing just by adding FCS and DMSO.

We want to encapsulate Islets of Langerhans in the capsules.

I am attempting isolation of islets from rat pancreas. I will be cutting the tissue (1x1mm) for collagenase digestion instead of purfusing collagenase into the pancreas. I would appreciate any suggestions for maximizing the yield, particularly about collagenase concentration, time of incubation etc.

Some studies proposed that corticosterone was inhibitory at earlier phase and enhances insulin secretion at late phase. Most of metabolic syndromes are involved with hyperaction of HPA axis, what will its effect be on insulin secretion?