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

IF 3.9 3区生物学 Q2 CELL BIOLOGY Mitochondrion Pub Date : 2024-05-06 DOI:10.1016/j.mito.2024.101890

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

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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.

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培养基的选择会影响 PBMC 定量、细胞大小和下游呼吸分析。

高分辨率呼吸测定法（HRR）可评估外周血单核细胞（PBMC）的生物能，但目前还没有用于制备 PBMC 和 HRR 分析的标准化培养基。在这里，我们研究了四种不同培养基（MiR05、PBS、RPMI、Plasmax）对完整 PBMC 细胞数量、大小和 HRR 分析（Oxygraph-O2k）的影响。值得注意的是，当 PBMC 重悬在 MiR05 中时，其细胞数比 PBS 或 Plasmax 中高 21%，从而导致 HRR 期间的氧气通量因固有调整而被低估。此外，在 MiR05 中观察到细胞体积变小和细胞聚集。根据我们的研究结果，我们认为 Plasmax、PBS 或 RPMI 比 MiR05 更适合用于完整 PBMC 的 HRR 分析。我们提供了 Plasmax 和 PBS 的氧溶解因子，并鼓励进一步优化完整 PBMC 的标准化 HRR 方案。

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来源期刊

Mitochondrion 生物-细胞生物学

CiteScore

9.40

自引率

4.50%

发文量

审稿时长

13.6 weeks

期刊介绍： Mitochondrion is a definitive, high profile, peer-reviewed international research journal. The scope of Mitochondrion is broad, reporting on basic science of mitochondria from all organisms and from basic research to pathology and clinical aspects of mitochondrial diseases. The journal welcomes original contributions from investigators working in diverse sub-disciplines such as evolution, biophysics, biochemistry, molecular and cell biology, genetics, pharmacology, toxicology, forensic science, programmed cell death, aging, cancer and clinical features of mitochondrial diseases.