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Methodology article
David Robert McIlwain
Stanford University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-2537-0273
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This work is licensed under a CC BY 4.0 License. Read Full License
Background: Cryopreserved 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.
Methods: Whole blood was processed in parallel using both Cell Preparation Tubesä (CPT, BD Biosciences) and Lymphoprepä 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 ten cell subsets, and capacity for stimulation-dependent CD4+ and CD8+ T cell intracellular cytokine production.
Results: No 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.
Conclusion: CPT and Lymphoprep tubes are effective and comparable methods for PBMC isolation for immunological studies.
Keywords
Peripheral blood mononuclear cells, BD Cell Preparation Tubes, Lymphoprep Tubes, flow cytometry, T cell, cytokine
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CURRENT STATUS: Under Review
Version 2
Posted 13 Feb, 2020
No community comments so far
Review #2 received
Received 29 Feb, 2020
Reviewer #2 agreed
On 15 Feb, 2020
Editor assigned
On 12 Feb, 2020
Reviewers invited
Invitations sent on 12 Feb, 2020
Reviewer #1 agreed
On 12 Feb, 2020
Submission checks complete
On 11 Feb, 2020
Editor invited
On 11 Feb, 2020
No comments provided
Editorial decision: Minor revision
On 04 Feb, 2020
Review #2 received
Received 03 Feb, 2020
Review #1 received
Received 03 Feb, 2020
Reviewer #3 agreed
On 21 Jan, 2020
Reviewer #2 agreed
On 20 Jan, 2020
Submission checks complete
On 17 Jan, 2020
Editor invited
On 17 Jan, 2020
Editor assigned
On 17 Jan, 2020
Reviewers invited
Invitations sent on 17 Jan, 2020
Reviewer #1 agreed
On 17 Jan, 2020
First submitted
On 16 Jan, 2020
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Subject Areas
Immunology
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This preprint is under consideration at BMC Immunology. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Methodology article
David Robert McIlwain
Stanford University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-2537-0273
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 2
Posted 13 Feb, 2020
No community comments so far
Review #2 received
Received 29 Feb, 2020
Reviewer #2 agreed
On 15 Feb, 2020
Editor assigned
On 12 Feb, 2020
Reviewers invited
Invitations sent on 12 Feb, 2020
Reviewer #1 agreed
On 12 Feb, 2020
Submission checks complete
On 11 Feb, 2020
Editor invited
On 11 Feb, 2020
No comments provided
Editorial decision: Minor revision
On 04 Feb, 2020
Review #2 received
Received 03 Feb, 2020
Review #1 received
Received 03 Feb, 2020
Reviewer #3 agreed
On 21 Jan, 2020
Reviewer #2 agreed
On 20 Jan, 2020
Submission checks complete
On 17 Jan, 2020
Editor invited
On 17 Jan, 2020
Editor assigned
On 17 Jan, 2020
Reviewers invited
Invitations sent on 17 Jan, 2020
Reviewer #1 agreed
On 17 Jan, 2020
First submitted
On 16 Jan, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Immunology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Cryopreserved 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.
Methods: Whole blood was processed in parallel using both Cell Preparation Tubesä (CPT, BD Biosciences) and Lymphoprepä 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 ten cell subsets, and capacity for stimulation-dependent CD4+ and CD8+ T cell intracellular cytokine production.
Results: No 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.
Conclusion: CPT and Lymphoprep tubes are effective and comparable methods for PBMC isolation for immunological studies.
Figure 1
Figure 2
Figure 3
Figure 4
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