From primary cells cultures of the human pancreas, we are getting positive immunostaining of cells for anti-insulin and anti-glucagon. We also get positive results from RT-PCR using a primer for insulin. I would feel better about our results if we had a definite positive control. I'd appreciate any comments at all about this.
I'm using an entire pancreas as a positive control, but I think that a cell line would be more comparable. Also, is there a human organ that would be absolutely negative for insulin?
Pancreatic Beta Cell Lines and their
Applications in Diabetes Mellitus Research
Masa Skelin , Marjan Rupnik and Avrelija Cen
Please read the article to find a table on insulin positive cell lines.
MIN6 is a cell line insulin+, but your positive control is the best you can have (entire pancreas), negative for insulin?, you can try skin keratinocytes
can I ask you something? wich antibodies are you using for anti-insulin and anti-glucagon (manufacturer)? Thanks!!
Pancreatic Beta Cell Lines and their
Applications in Diabetes Mellitus Research
Masa Skelin , Marjan Rupnik and Avrelija Cen
Please read the article to find a table on insulin positive cell lines.
Reported insulin-producing
cell lines such as RINm5F, RINr , NIT-1
, flTC-1, flTC-3, IgSV195 , and HIT cells do not respond to glucose, or respond at much lower glucose concentrations (1 to 5 mmol/l) than do normal islets. Although a recently developed cell line, INS-l,
seemed to respond to the physiological range of glucose,
only a 2.2-fold increase in insulin secretion was observed
at a high glucose concentration [11]. Abnormal properties
of glucose transport and/or phosphorylation have been reported
for RINm5 F, RINr, flTC-1,
flTC-3, IgSV195, and HIT cells, leading to
characteristics of glucose utilization which differ from
those of normal pancreatic beta cells.
For the comprehensive study u cAN ALSO REFER THIS REVIEW ARTICLE.....
"Pancreatic Beta Cell Lines and their Applications in Diabetes Mellitus Research"
I THINK THIS WILL GREATLY HELP U IN CHOOGING UR CELL LINE OF INTEREST
Most beta cell lines are derived from rodents (like MIN6, INS-1, RIN). However, a stable functional HUMAN beta cell line was described in:
Ravassard P, Hazhouz Y, Pechberty S, Bricout-Neveu E, Armanet M, Czernichow P, Scharfmann R. A genetically engineered human pancreatic beta cell line exhibiting glucose-inducible insulin secretion. J Clin Invest 2011;121:3589-97.
Thank you everyone! I have plenty to look into here. @ Bernard: I'm using the Abcam anti-INS and anti-GCG.
Hey !! Can you tell me that what is the procedure you are following for primary culture of pancreas ? Are you using whole organ or isolating the islets from it ?
Could you please post here the link of article you are following !! Thank you !!
You can use the different insulinom cell lines most preferably rin 5f, min 6, ins-1 !!
Hope ! It would be helpful .
Ritu: I am not following anyone else's protocol--this is all new to our lab. We're macerating the pancreas to a single-cell suspension, then culturing in flasks and on coverslips in DMEM-10% FBS-1% antimicrobials. It's difficult to get a single cell suspension. I've tried 0.1% collagenase, next I'm going to try trypsin.
Patrick, thank you. I'm having trouble getting the supplemental materials link to open, but will continue to try.
Our lab collaborates with some of the folks there at UofA, that were originally in Bernard Thebaud's lab--and still collaborate with him in Ottawa as well. We've been sending them human fetal lung tissue for years.
MIN6 or nothing
only ins positive
no glucagon
tricky to keep going
form islet like structures
expressed connexin36
Just a note that the stable functional HUMAN beta cell line that was described in:
Ravassard P, Hazhouz Y, Pechberty S, Bricout-Neveu E, Armanet M, Czernichow P, Scharfmann R. A genetically engineered human pancreatic beta cell line exhibiting glucose-inducible insulin secretion. J Clin Invest 2011;121:3589-97 are 2000 Euros plus shipping as part of their MTA
Concerning the second part of your question, insulin is present in the pancreas, the thymus and the brain in the course of development. But the highest expression is indeed in the adult pancreas. Experiments in rodents show insulin expression in the liver and in lacrymal glands following prolonged hyperglycemia. So for your negative control, you can better use several other organs than these ones, collected from a normoglycemic individual.
With regard to the first question, I don't bring a solution, but a clarification. You are working apparently with dissociated "total pancreas", if I get correctly your reply to Ritu. It is therefore logic that you detect insulin/glucagon because they are there among the dissociated cells. But this is a tricky business because even if you fish the islets (what we do in our group), you can't get all endocrine cells (islets fractions) out of your sample. So there will always be some ins/gcg signal in your prep. Unfortunately, there is no known toxin for these human cells as is the case with rodent beta cells that are sensistive to Alloxan or Streptozotocin. This should be taken into account when analysing your data!
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