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

Area of Science:

• Cellular bioenergetics
• Respirometry techniques
• Immunology

Background:

• High-resolution respirometry (HRR) is vital for assessing cellular energy metabolism.
• Standardized protocols for preparing peripheral blood mononuclear cells (PBMCs) for HRR are lacking.
• The choice of respirometry medium can significantly impact cell count, size, and bioenergetic measurements.

Purpose of the Study:

• To investigate the impact of four different media (MiR05, PBS, RPMI, Plasmax) on PBMC preparation and subsequent HRR.
• To determine the optimal medium for accurate assessment of intact PBMC bioenergetics using HRR.
• To provide data supporting the development of a standardized HRR protocol for PBMCs.

Main Methods:

• Intact PBMCs were prepared and analyzed in four different media: MiR05, PBS, RPMI, and Plasmax.
• PBMC count and size were determined for each medium.
• Oxygen (O2) flux was measured using high-resolution respirometry (HRR) with the Oxygraph-O2k instrument.
• Oxygen solubility factors for Plasmax and PBS were determined.

Main Results:

• Resuspending PBMCs in MiR05 resulted in a 21% higher cell count compared to PBS or Plasmax.
• MiR05 medium led to smaller cell size and increased cell aggregation.
• Higher cell counts in MiR05 caused an underestimation of O2 flux during HRR.
• Plasmax, PBS, and RPMI were found to be more suitable than MiR05 for HRR of intact PBMCs.

Conclusions:

• The choice of medium significantly affects PBMC count, size, aggregation, and subsequent HRR measurements.
• MiR05 is not recommended for HRR of intact PBMCs due to its impact on cell count and O2 flux.
• Plasmax, PBS, or RPMI are suggested as more appropriate media for standardized HRR protocols of intact PBMCs.
• Further optimization and standardization of HRR protocols for intact PBMCs are encouraged.