Keppner 2018 MiP2018
Peripheral Blood Mononuclear Cells (PBMCs) are a collection of different blood cells with the similarity to have a round nucleus. These include myeloid cells such as Monocytes (10-29%) and Dendritic cells (1-2%) as well as lymphoid cells particularly B cells (10-15%), T cells (50-80%), and NK cells (10-15%). Since PBMCs are easily accessible by isolating them from a blood sample, they serve as an ideal source to study mitochondrial function of human cells. Right now PBMCs have to be measured immediately after isolating them from the whole blood, according to the standard protocol for mitochondrial respiration of PBMCs. Collecting many blood samples on the same day is not possible since there is only a short time-window during which mitochondrial activity can be assessed. In the present project a protocol was established for the cryopreservation and thawing of PBMCs for high-resolution respirometry. Consequently, the effect of cryopreservation was analyzed regarding the mitochondrial function of PBMCs. Whole blood was obtained by venipuncture with Lithium-Heparin Monovettes to prevent blood clotting and PBMCs were isolated by density gradient centrifugation. Freshly isolated PBMCs were either cryopreserved at – 80°C for at least 7 days or directly measured by high-resolution respirometry.
Our results show comparable respiration rates of freshly isolated and cryopreserved PBMCs. It is possible to cryopreserve PBMCs under optimal conditions, which means to thaw quickly, dilute slowly and pipette very gently. Otherwise cryopreserved PBMCs show a reduced uncoupled respiration under FCCP, which can also be prevented by adding Cytochrome C. We present here a possibility to cryopreserve PBMCs for high resolution respirometry.
Keppner G(1,2), Bayer F(1), Skurk T(2,3), Klingenspor M(1,2)
- Chair Molecular Nutritional Medicine, Technische Univ München, Else Kröner-Fresenius Zentrum Ernährungsmedizin. - [email protected]
- ZIEL– Inst Food and Health, Technische Univ München
- Chair Clinical Nutritional Medicine, Technische Univ München, Else Kröner-Fresenius Zentrum Ernährungsmedizin
Labels: MiParea: Respiration, Instruments;methods
Tissue;cell: Blood cells Preparation: Intact cells
Coupling state: ET