Abstract
BackgroundCryopreserved human peripheral blood mononuclear cells (PBMCs) are a commonly used sample type for a variety of immunological assays. Many factors can affect the quality of PBMCs, and careful consideration and validation of an appropriate PBMC isolation and cryopreservation method is important for well-designed clinical studies. A major point of divergence in PBMC isolation protocols is the collection of blood, either directly into vacutainers pre-filled with density gradient medium or the use of conical tubes containing a porous barrier to separate the density gradient medium from blood. To address potential differences in sample outcome, we isolated, cryopreserved, and compared PBMCs using parallel protocols differing only in the use of one of two common tube types for isolation.MethodsWhole blood was processed in parallel using both Cell Preparation Tubes™ (CPT, BD Biosciences) and Lymphoprep™ Tubes (Axis-Shield) and assessed for yield and viability prior to cryopreservation. After thawing, samples were further examined by flow cytometry for cell yield, cell viability, frequency of 10 cell subsets, and capacity for stimulation-dependent CD4+ and CD8+ T cell intracellular cytokine production.ResultsNo significant differences in cell recovery, viability, frequency of immune cell subsets, or T cell functionality between PBMC samples isolated using CPT or Lymphoprep tubes were identified.ConclusionCPT and Lymphoprep tubes are effective and comparable methods for PBMC isolation for immunological studies.
Highlights
Cryopreserved human peripheral blood mononuclear cells (PBMCs) are a commonly used sample type for a variety of immunological assays
Viability, frequencies of 10 unique immune subsets, and cytokine production in both CD4+ and CD8+ T cells in response to two stimulation conditions; each measurement showing no significant differences between the Cell Preparation Tubes (CPT) and LP tube methods. These results indicate that CPT and LP tubes are suitable for collection of high-quality PBMCs for downstream immunological assays
PBMC recovery and viability are comparable between CPT and Lymphoprep methods Two PBMC processing and cryopreservation methods were designed differing only in use of CPT or LP tubes
Summary
Cryopreserved human peripheral blood mononuclear cells (PBMCs) are a commonly used sample type for a variety of immunological assays. A major point of divergence in PBMC isolation protocols is the collection of blood, either directly into vacutainers pre-filled with density gradient medium or the use of conical tubes containing a porous barrier to separate the density gradient medium from blood. The central principle of PBMC isolation protocols is centrifugal separation of blood components against a high-density medium such as Ficoll-Paque®, Histopaque®-1077, etc. The first option is to draw blood directly into a vacutainer containing Ficoll-Paque® density gradient medium separated from blood by a thick layer of polyester resin such as in Cell Preparation Tubes (CPT) manufactured by BD Biosciences. The second option is to load diluted blood into tubes containing a high-density gradient medium separated by a plastic barrier (e.g. frit). Plastic barrierbased products include tubes that come pre-filled with high-density separation media, such as Lymphoprep Tubes (Axis-Shield) and EZ Lympho-SepTM Lymphocyte Separation Tubes (Biological Industries), or those that contain only the polyethylene insert, such as SepMateTM Tubes (STEMCELL Technologies) and AccuspinTM Tubes (Sigma-Aldrich)
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