I would like to take rat kidney tissue and isolate from that the proximal tubule cells for immediate use in a Clark-type oxygen electrode to study mitochondrial respiration. I do not want to study isolated mitochondria (for reasons I will not go into here), and using tissue sections (e.g. 200 micron thin slices) does not allow full respirometry studies to be performed, only crude O2 consumption rates (even with permeabilisation).
I have seen lots of protocols for isolating PTCs for use then as primary cell lines however I will not be using the cells for this purpose. In my experiments the cells will be discarded after the short respirometry studies have been completed. I'm unsure of how much of these protocols I should follow if I just want to obtain a cell suspension for immediate use and not prolonged culture. Ideally I would like something quicker and simpler than most of the protocols I have found so far seem to be.
Prior to respirometry studies the naive kidney tissue will have been sliced to 200 um thick and exposed to certain insults (e.g. LPS, septic serum, etc.) for around 90 min. I would like a method that is as quick as possible to get the PTCs from the tissue in a state in which I can add them to the Clark electrode. This is in order to observe any acute changes that may have occurred to the mitochondrial function during incubation which may also revert relatively quickly once removed from the insulting environment. Once I have a cell suspension I intend to permeabilise to allow addition of the respiratory substrates, ADP, uncouplers, etc. required for respirometry studies.
If it's likely that this potential method will encounter too many problems I may first isolate the PTCs and then expose them to my various insults immediately before washing and beginning the respirometry studies. In that case would I need to culture the cells, or could I just expose them in suspension? Ideally I would however like to be able to expose slices to the insult (for reasons of consistency with other methodologies I am currently employing) as initially suggested.
Many thanks.
Hi
this link can help you
https://www.sciencellonline.com/products-services/primary-cells/rat-renal-proximal-tubular-epithelial-cells.htm
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066750
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