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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol presents an accessible guide for collecting, storing, and thawing peripheral blood mononuclear cells suitable for downstream analyses and workflows like flow cytometry and RNA sequencing. Plasma and buffy coat collections are also demonstrated.

Abstract

Peripheral blood mononuclear cells (PBMCs) are commonly used in biomedical research on the immune system and its response to disease and pathogens. This detailed protocol describes the equipment, supplies, and steps for isolating, cryopreserving, and thawing high-quality and highly viable PBMCs from whole blood cells suitable for downstream applications such as flow cytometry and RNA-sequencing. Protocols for processing plasma and buffy coat from the whole blood in parallel and concurrently with PBMCs are also described. This easy-to-follow step-by-step protocol, which utilizes density gradient centrifugation to isolate PBMCs, is accompanied by a checklist of supplies, equipment, and preparation steps. This protocol is suitable for individuals with any prior experience with laboratory techniques and can be implemented in clinical or research laboratories. High-quality cell viability and RNA sequencing resulted from PBMCs collected by operators with no prior laboratory experience using this protocol.

Introduction

This protocol demonstrates an accessible method and workflow for the isolation of peripheral blood mononuclear cells (PBMCs) from whole blood. This protocol is especially targeted towards novice research technicians, students, and clinical lab staff with the objective of facilitating the collection and cryopreservation of PBMCs without assuming prior training in laboratory techniques.

This protocol utilizes centrifugation to separate the components of whole blood by density. Whole blood consists of four main components listed in order of decreasing density: red blood cells/erythrocytes (~45% of volume), white blood cel....

Protocol

The study protocol was approved by the UCSD and KUMC Human Protections Program and conforms to the Declaration of Helsinki. All individuals provided informed consent for participation and blood collection.

1. Processing and cryopreservation of PBMCs

  1. One day prior to the blood collection, print out the checklist of materials and reagents listed in Table 2 and prepare and label accordingly.
    NOTE: This protocol was designed for the collection o.......

Representative Results

Following PBMC collection and cryopreservation, viability of thawed PBMCs, monocytes, and lymphocytes from 56 unique samples was assessed by flow cytometry using reagents listed in Table 4 following manufacturers' instructions (Figure 2A-F). A mean ± SD viability of PBMCs, monocytes, and lymphocytes of 94 ± 4.0%, 98 ± 1.1%, and 93 ± 5.6%, respectively, was achieved (Figure 2G). Viability measurement by Tr.......

Discussion

This protocol for PBMC collection and cryopreservation has been successfully implemented by individuals with and without prior research laboratory training. In our application, FACS and RNA-sequencing of highly viable monocytes purified from stored PBMCs resulted in high-quality sequences.

A major strength of this protocol is its accessibility. The technique presented in the protocol utilizes tubes pre-packed with a solid-density gradient medium. As a result, whole blood can be collected direc.......

Acknowledgements

We'd like to thank the patients who volunteered their consent, time, and donation of blood samples. We also acknowledge Dr. Patrick Moriarty, Julie-Ann Dutton, and Mark McClellen at the Kansas University Medical Center for their collaboration and for implementing this protocol at a remote site. CY has received research support from NIH grant 1K08HL150271.

....

Materials

NameCompanyCatalog NumberComments
1000 µL TipsGilsonF174501Approximately 3 tips needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
15 mL tubeBiopioneeerCNT-15To hold Solutions 2/3
2 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
2 mL CryovialsGlobe Scientific3012Approximately 40 cryovials needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
20 µL TipsGilsonF174201For use in cell counting; volume needed is dependent on method of cell counting used. 1 tip is needed per 1 mL of unpooled frozen PBMCs to be defrosted
50 mL Conical TubeCEM Corporation50-187-76832 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
3 needed per 1 mL of unpooled frozen PBMCs to be defrosted
CD14 AntibodyBiolegend325621
CD16 AntibodyBiolegend360723
CD19 AntibodyBiolegend363007
CD3 AntibodyBiolegend300405
CD56 AntibodyBiolegend318303
CD66b AntibodyBiolegend305103
Cell StrainerBiopioneeerDGN2583671 needed per 1 mL of unpooled frozen PBMCs to be defrosted
CPT Mononuclear Cell Preparation TubeBD Biosciences3627536 tubes needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Cryo Freezer BoxSouthern LabwareSB2CC-81Holds 81 tubes
1 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Cryotube RackFisherbrand05-669-45Holds up to 50 cryovials
DMSOInvitrogen15575020Approximately 2.5 mL needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
DNase/RNase-Free Distilled WaterInvitrogen10977015Approximately 2 mL needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Approximately 9 mL needed per 1 mL of frozen PBMCs to be defrosted
EDTA TubesBD Biosciences3666431 tube needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
FlavopiridolSigma-AldrichF3055-5MG
HLA-DR AntibodyBiolegend307609
Human Serum AlbuminGeminiBio800-120Approximately 25 mL needed per patient or treatment condition for PBMC/Plasma/Buffy Coat Collection
Human Trustain FcXBiolegend422301
IsopropanolSigma-AldrichW292912-1KG-KFor use in Mr. Frosty
Label PrinterPhomemoM110-WH
Live-Dead StainBiolegend423105
Mr. FrostyThermo Scientific5100-0001Holds up to 18 tubes.
2 Mr. Frostys needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Multidispense PipetteBrandtech705110
Multidispense Pipette TipsBrandtech705744Approximately 3 tips needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
P1000 PipetteGilsonF144059M
P20 PipetteGilsonF144056MFor use in cell counting; volume needed is dependent on method of cell counting used.
Printer LabelsPhomemoPM-M110-3020Approximately 45 labels needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Approximately 5 labels needed per 1 mL of unpooled frozen PBMCs to be defrosted
RNase-Free EDTA (0.5 M)InvitrogenAM9260GApproximately 40 µL needed per 1 mL of frozen PBMCs to be defrosted
RNase-Free PBS (10X)InvitrogenAM9625Approximately 1 mL needed per 1 mL of frozen PBMCs to be defrosted
RNasinPromegaN2111
RPMICorning10-040-CVApproximately 80 mL needed per patient or treatment condition for PBMC/Plasma/Buffy Coat Collection
Transfer PipettesFisherbrand13-711-7MApproximately 5 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Tube HolderEndicott-Seymour14-781-15Holds up to 80 CPT/EDTA Tubes

References

  1. Mathew, J., Sankar, P., Varacallo, M. . Physiology, Blood Plasma. , (2018).
  2. Van Oost, B., Van Hien-Hagg, I. H., Timmermans, A. P., Sixma, J. J. The effect of thrombin on the density distribution of blood platelets: Detection of activate....

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