Brianna A Werner1,2, Patrick J McCarty1,2, Alison L Lane1,2, Indrapal Singh1,2, Mohammad A Karim1,2, Shannon Rose3, Richard E Frye1,2. 1. Section on Neurodevelopmental Disorders, Department of Neurology, Barrow Neurological Institute at Phoenix Children's Hospital Phoenix, AZ, USA. 2. Department of Child Health, University of Arizona College of Medicine - Phoenix Phoenix, AZ 85016, USA. 3. Arkansas Children's Research Institute Little Rock, AR 72202, USA.
Abstract
OBJECTIVES: Bioenergetic measurements in peripheral blood mononuclear cells (PBMCs) using high-throughput respirometry is a promising minimally invasive approach to studying mitochondrial function in humans. However, optimal methods for collecting PBMCs are not well studied. METHODS: Bioenergetics and viability were measured across processing delays, tube type and cryopreservation. RESULTS: Storage of collection tubes on dry ice resulted in unrecoverable samples and using the Cell Preparation Tube (CPTTM) significantly reduced viability. Thus, storage in Sodium Citrate (NaC) and ethylenediaminetetraacetic acid (EDTA) tubes were studied in detail. Cell viability decreased by 0.5% for each hour the samples remained on wet ice prior to processing while cryopreservation decreased viability by 9.6% with viability remaining stable for about one month in liquid nitrogen. Adenosine triphosphate linked respiration (ALR) and proton-leak respiration (PLR) changed minimally while maximal respiratory capacity (MRC) and reserve capacity (RC) decreased markedly with collection tubes stored on wet ice over 24 hrs. Changes in respiratory parameters were more modest over the first 8 hours. Manipulations to replace media did not attenuate changes in respiratory parameters. Cryopreservation decreased ALR, MRC and RC by 17.20, 95.30 and 54.92 pmol/min, respectively and increased PLR by 2.65 pmol/min. PLR, MRC and RC changed moderately during the first month in liquid nitrogen for freshly frozen PBMCs. CONCLUSIONS: Our results suggest that bioenergetics in PBMCs vary based on the processing time from specimen collection and preservation method. Changes in bioenergetics can be minimized by processing samples with a minimal time delay. Changes in viability are minimal and may not correspond to changes in bioenergetics. AJTR
OBJECTIVES: Bioenergetic measurements in peripheral blood mononuclear cells (PBMCs) using high-throughput respirometry is a promising minimally invasive approach to studying mitochondrial function in humans. However, optimal methods for collecting PBMCs are not well studied. METHODS: Bioenergetics and viability were measured across processing delays, tube type and cryopreservation. RESULTS: Storage of collection tubes on dry ice resulted in unrecoverable samples and using the Cell Preparation Tube (CPTTM) significantly reduced viability. Thus, storage in Sodium Citrate (NaC) and ethylenediaminetetraacetic acid (EDTA) tubes were studied in detail. Cell viability decreased by 0.5% for each hour the samples remained on wet ice prior to processing while cryopreservation decreased viability by 9.6% with viability remaining stable for about one month in liquid nitrogen. Adenosine triphosphate linked respiration (ALR) and proton-leak respiration (PLR) changed minimally while maximal respiratory capacity (MRC) and reserve capacity (RC) decreased markedly with collection tubes stored on wet ice over 24 hrs. Changes in respiratory parameters were more modest over the first 8 hours. Manipulations to replace media did not attenuate changes in respiratory parameters. Cryopreservation decreased ALR, MRC and RC by 17.20, 95.30 and 54.92 pmol/min, respectively and increased PLR by 2.65 pmol/min. PLR, MRC and RC changed moderately during the first month in liquid nitrogen for freshly frozen PBMCs. CONCLUSIONS: Our results suggest that bioenergetics in PBMCs vary based on the processing time from specimen collection and preservation method. Changes in bioenergetics can be minimized by processing samples with a minimal time delay. Changes in viability are minimal and may not correspond to changes in bioenergetics. AJTR
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