I am currently working on characterization of lymphocytes extracted from diseased kidney tissue (i.e. acute rejection). Until now the piece of kidney that we received was big enough to extract lymphocytes just by mechanical dissection, but in the next weeks we will receive small biopsies and I am not sure if mechanical dissection will be enough. Also, I have read that with enzymatic digestion the surface markers can be disrupted by the collagenase (and I need them for further FACS analysis). Any suggestion?
Thank you in advance!
Hi Raul,
A combination of mechanical and chemical dissociation works best, usually. I wouldn't use trypsin because of the surface markers, as you said. Other proteases that are more specific like accutase or collagenase should be fine for most markers though. Sometimes EDTA alone can do wonders to open up adherens junctions.
- Hendrik
Dear Raul,
I agree with Hendrik. And, you can use a Medimachine device for mechanical dissociation.
Thank you very much for your answers. For the first samples we will try mechanical dissociation manually and then probably collagenase IV, that has very low concentration of trypsin. If it works out we will think about the Medimachine.
Ishak, can I ask you if you have used before the Medimachine for lymphocyte isolation from tissue or if you know any specific publication related?
Thank you in advance, and I will describe the results after the first try.
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