Choice of medium affects PBMC quantification, cell size, and downstream respiratory analysis

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High-resolution respirometry (HRR) can assess peripheral blood mononuclear cell (PBMC) bioenergetics, but no standardized medium for PBMC preparation and HRR analysis exist. Here, we study the effect of four different media (MiR05, PBS, RPMI, Plasmax) on the count, size, and HRR (Oxygraph-O2k) of intact PBMCs. Remarkably, the cell count was 21 % higher when PBMCs were resuspended in MiR05 than in PBS or Plasmax, causing O2 flux underestimation during HRR due to inherent adjustments. Moreover, smaller cell size and cell aggregation was observed in MiR05. Based on our findings, we propose that Plasmax, PBS or RPMI is more suitable than MiR05 for HRR of intact PBMCs. We provide oxygen solubility factors for Plasmax and PBS and encourage further optimization of a standardized HRR protocol for intact PBMCs.

OriginalsprogEngelsk
Artikelnummer101890
TidsskriftMitochondrion
Vol/bind77
Antal sider7
ISSN1567-7249
DOI
StatusUdgivet - 2024

Bibliografisk note

Funding Information:
The authors gratefully acknowledge the Core Facility for Integrated Microscopy at University of Copenhagen, PhD Christophe C\u00F4me from Biotech Research & Innovation Centre and Epigenomlab, University of Copenhagen, Laboratory technicians Kristoffer Racz from Department of Biomedical Sciences, University of Copenhagen, as well as Sofie Lindegaard Jakobsen and Jonathan Wolters from Department of Biology, University of Copenhagen, for their support and excellent technical assistance in this work. This work was supported by Danish Diabetes Academy, Denmark [grant number NNF17SA0031406; LG]; Fru Astrid Thaysens Legat for L\u00E6gevidenskabelig Grundforskning, Denmark; Dagmar Marshalls Fond, Denmark; and the Independent Research Fund Denmark [grant number DFF-8022-00301B; MK].

Funding Information:
This work was supported by Danish Diabetes Academy [grant number NNF17SA0031406 ; LG], Fru Astrid Thaysens Legat for L\u00E6gevidenskabelig Grundforskning, Dagmar Marshalls Fond , and the Independent Research Fund Denmark [grant number DFF-8022-00301B ; MK].

Publisher Copyright:
© 2024 The Author(s)

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