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.
An overview of publications using the O2k with blood cells in general can be found when using our MiPap O2k-Publications:_Topics and selecting "Blood cells" in the Tissues, cell types and cell lines section O2k-Publications:_Blood_cells.
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]]
Will Kotiadis
As MiRO is essentially a cytosol mimic medium it contains high levels of certain ions (i.e. K, potassium) that may affect physiological function under such conditions. I would reccomend that you use the same medium you use for culturing your cells and replace the carbonate buffer with HEPES (Must be careful not to just add HEPES to medium containing carbonate as this will also affect buffering capacity). If this doesn`t work it may be that your respiratory capacity under such growth conditions is minimal, in which case you may wish to enhance it by replacing glucose in the medium with galactose. This should lead to a substantial increase in Respiratory capacity in most cell types. I hope this help! [[email protected]]
Erika Cortez
I published a paper last year with lymphocytes isolated from blood, measuring their respiratory capacity using oxygraph. I used the MIR05 medium and also used digitonin in order to permeabilize the mito membrane. The respiratory flux of these cells is really very low, so you need to set the O2 flux scale (in the graphic) from 0 until 20 (pmols O2.s-1.million cells-1) to observe differences between respiratory states.
ScientificWorldJournal. 2012;2012:629326. doi: 10.1100/2012/629326. Epub 2012 Mar 12. Lymphocytes mitochondrial physiology as biomarker of energy metabolism during fasted and fed conditions. Cortez E, Neves FA, Bernardo AF, Stumbo AC, Carvalho L, Garcia-Souza E, Sichieri R, Moura AS. [[email protected]]
