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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:, and more recently here: 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: (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 O2k:
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. - [[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)<> or myself magnus.hansson (at)<> - [[email protected]]

Will Kotiadis

As MiR05 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]]

MiPNet discussion forum: lymphocyte isolation for respirometry (2013-03-31)

Marcus F Oliveira

We are planning to investigate mitochondrial physiology on intact human blood derived lymphocytes and monocytes by HRR-SUIT protocols. We are experiencing problems regarding the detection of oxygen fluxes on both intact cells after cell sorting by flow cytometry. In fact, we were unable to detect any oxygen consumption at all after isolation. We though on buffer composition during cell sorting but we eventually knew that this was not the case. We have found a recent work from Dr. Victor Darley-Usmar group (Chacko et al., Life Sci, 2013), but they perform the sorting using magnetic beads, which will not be applied in our case.
Does anyone know a method to isolate these cells by cell sorting for proper respirometry analyses ?

Nicole Van Bergen

We routinely measure respiration in lymphoblasts but I don't see why the same protocols cannot apply to lymphocytes. We have very recently published full laboratory protocols for this isolation, as well as subsequent respiration protocols in Mitochondrion ( The supplementary material contains full laboratory protocols as well as guidelines for protocol optimisation. Blood needs to be freshly collected in blood tubes with K-EGTA to prevent coagulation, always kept at room temperate (never refrigerated), blood is then diluted with cell culture media (no serum) or buffered salt solutions and separated on ficoll-paque gradients.
We have used these techniques to detect differences in respiration of glaucoma patients:

Walter E Mueller

Please see our paper [Leuner et al Mol.Neurobiol. 46, 194-204, 2012]. Prof. Eckert Basel was responsible for the respirometry data.

Jelle Achten

In our lab we have done a lot of measurements on fibroblasts and lymphocytes. For both materials we used the Mir05 medium and we did the isolation for the lymphocytes with Lymphoprep. This is a kind of ficoll paque gradient we use in our lab with a special collect tube with a filter. The art. Nr for these disposables are down below. For preparation of the blood I agree with Nicole Van Bergen. Very important factors are fresh blood (not older then 48h), keep at room temperature and K-EGTA blood tubes to prevent coagulation. We also did a cell differentiation to have a look at the % of lymphocytes and monocytes, were we noticed the freshness is very important.
Leucosep Tubes: Greiner 163289
Lymphoprep: Axis Shield 1114545