Dear all,
I'm isolating pancreatic islets from human donor pancreas.
After the organ's perfusion and digestion (1 mg/ml Collagenase P, until free islets are visible with DTZ staining) and after centrifuging the digested (400xg, 2 minutes, +4°C), when I resuspend the pellet in HBSS complemented with 2% Serum, the pellet forms filaments that I can not break up and most of the islets remain trapped in these structures.
Has it ever happened to anyone?
How can I avoid the formation of these filaments?
Thank you very much.
Hi Nicola,
I don't think I've ever seen anything like that. Are you using human serum? Would you say these filaments are "sticky"?
Thanks,
Paul
Hi Paul,
Thank you very much.
in these first attempts I am not using human serum. Do you think this may be the problem?
Yes, filaments are "sticky".
Thanks,
Nicola
Hi again,
The "stickiness" may come from DNA from ruptured cells. You may need to use pulmozyme to cleave the DNA.
Thanks,
Paul
Can you suggest me a type of pulmozyme?
What concentration of pulmozyme is used?
Try looking at these: Article Adjustment of digestion enzyme composition improves islet is...
Article Human Pancreatic Islet Isolation: Part I: Digestion and Coll...
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