I tried to digest the liver with a collagenase/DNase protocol but it didn't work. I also tried a mechanical digestion but I have too much fragments. Could anyone help me?
Thank you
We don't isolate cells regularly from adult liver, so i'm by no means an expert, but I've had okay luck mechanically dissociating liver using a dounce pestle b. Just resuspend in PBS or any old buffer and dounce 10-15 times. If you intend to do facs you'll want to strain the cells through a filter so the machine won't clog. There are probably much better ways but that's what I tried.
Thank you for your answer. But that's what I tried and it didn't work.. When I did the FACS analysis, only 1% of the suspension was entire cells. But thank you!
I think the problem here is that you are working with a dense/compact tissue, I doubt you would ever get all the cells, 1% might actually be a good starting point. The real questions are: What are you looking for? Would microscopy be a better tool? Can you pre-treat (cut out the bits you need)? Can you culture the cells of interest? Normally, (in my experience) the choice tissues for maceration are lymph nodes/spleens/bone marrow, even brain. All these tissues much less dense than liver.
Thank you for your answer. I tried to get the percentage of immune cells vs hepatic cells. But I think I'm gonna go with microscopy, even if it's not really quantitative.
Not sure if this will work.... But it might be worth a try. When we make spleen single cell suspensions we use the sterilized frosted glass slide technique. The tissue is placed between the frosted portion of the 2 slides and quickly and carefully "lightly ground" into a single cell suspension. It is very important to use a Ca++/Mg++ FREE item such as HBSS or PBS with 2%HI FBS to keep the cells happy and from clumping.
Please let me know if you try this, and if it works.
Thanks, Robert
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