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

Ida Bager Christensen; Lucas Ribas; Maria Mosshammer; Marie Louise Abrahamsen; Michael Kühl; Steen Larsen; Flemming Dela; Linn Gillberg

doi:10.1016/j.mito.2024.101890

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

Ida Bager Christensen, Lucas Ribas, Maria Mosshammer, Marie Louise Abrahamsen, Michael Kühl, Steen Larsen, Flemming Dela, Linn Gillberg^*

^*Corresponding author for this work

Research output: Contribution to journal › Journal article › Research › peer-review

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Abstract

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.

Original language	English
Article number	101890
Journal	Mitochondrion
Volume	77
Number of pages	7
ISSN	1567-7249
DOIs	https://doi.org/10.1016/j.mito.2024.101890
Publication status	Published - 2024

Bibliographical note

Publisher Copyright:
© 2024 The Author(s)

Keywords

Cell count
High-resolution respirometry
Medium
Oxygen solubility
Peripheral blood mononuclear cells

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10.1016/j.mito.2024.101890Licence: CC BY

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Choice of medium affects PBMC quantification, cell size, and downstream respiratory analysis. / Bager Christensen, Ida; Ribas, Lucas; Mosshammer, Maria ; Abrahamsen, Marie Louise ; Kühl, Michael ; Larsen, Steen ; Dela, Flemming ; Gillberg, Linn.

In: Mitochondrion, Vol. 77, 101890, 2024.

Research output: Contribution to journal › Journal article › Research › peer-review

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

abstract = "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.",

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author = "{Bager Christensen}, Ida and Lucas Ribas and Maria Mosshammer and Abrahamsen, {Marie Louise} and Michael K{\"u}hl and Steen Larsen and Flemming Dela and Linn Gillberg",

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T1 - Choice of medium affects PBMC quantification, cell size, and downstream respiratory analysis

AU - Bager Christensen, Ida

AU - Ribas, Lucas

AU - Mosshammer, Maria

AU - Abrahamsen, Marie Louise

AU - Kühl, Michael

AU - Larsen, Steen

AU - Dela, Flemming

AU - Gillberg, Linn

PY - 2024

Y1 - 2024

N2 - 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.

AB - 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.

KW - Cell count

KW - High-resolution respirometry

KW - Medium

KW - Oxygen solubility

KW - Peripheral blood mononuclear cells

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DO - 10.1016/j.mito.2024.101890

M3 - Journal article

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AN - SCOPUS:85193636163

VL - 77

JO - Mitochondrion

JF - Mitochondrion

SN - 1567-7249

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