Talk:Respirometry

MiPNet discussion forum: respiratory measurements with lymphocytes (2013-10-24)

Magda Labieniec-Watala

Lymphocytes and respiratory measurements - can anyone help? I have just started to work with lymphocytes isolated from blood and I am interested in measuring their respiratory capacity using oxygraph. I have used the MIRO6 medium but probably it was a mistake. Lymphocytes do not respire and do not respond to substrates and inhibitors in this environment. Thus, I have a request for experimental protocol for these measurements from those who study the mitochondrial respiratory in lymphocytes. May enyone do similar experiments and have an appopriate experience? I would like to add that we do not isolate mitochondria from lymphoctes but we use digitonin in order to permeabilize the mito membrane. I tried to find some papers concerning these studies, but without success. I would be very grateful for some suggestions in this issue. Thank you in advance. [[email protected]]

Gerhard Krumschnabel

Lymphoblasts have been examined in here: http://www.ncbi.nlm.nih.gov/pubmed/22427588, and more recently here: http://bioblast.at/index.php/Pecina_2013_Abstract_MiP2013. Generally, for experiments in permeabilized cells [you do not (and would not want to) permeabilize the mitochondrial membrane with digitonin, but the plasma membrane], you need to optimize conditions; I suggest you follow the protocol given in this reference: http://bioblast.at/index.php/Pesta_2012_Methods_Mol_Biol (download "Bioblast pdf"). Further, there is a recent paper (using our competitor's equipment - but, so what?) examining respiration in INTACT blood cells (platelets, lymphocytes, monocytes, neutrophils), at least this should be easily possible with an oxygraph: http://www.ncbi.nlm.nih.gov/pubmed/23528848.

Marcus Oliveira

I have previously worked with human PBMCs and made respiratory analyses on permabilized cells using succinate and rotenone (see paper attached). Maybe this could help you on your experiments. http://www.ncbi.nlm.nih.gov/pubmed/21336129 [[email protected]]

Magnus Hansson

We have recently published study of Peripheral blood mononuclear cells (mostly lymphocytes after preparation) in sepsis. See if you can find useful information there, otherwise feel free to contact Fredrik.Sjovall (at) med.lu.se<http://med.lu.se> or myself magnus.hansson (at) med.lu.se<http://med.lu.se> http://www.ncbi.nlm.nih.gov/pubmed/23883738 [[email protected]]