Isolation, Cryopreservation, and Immunophenotyping of Human Peripheral Blood Mononuclear Cells

Fredine T. Lauer1, Jesse L. Denson1, Scott W. Burchiel1

1 Department of Pharmaceutical Sciences, The University of New Mexico College of Pharmacy, Albuquerque
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.20
DOI:  10.1002/cptx.31
Online Posting Date:  November, 2017
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Abstract

This unit describes procedures for the isolation, cryopreservation, and thawing of human peripheral blood mononuclear cells (HPBMC) and analysis of cell surface markers (CSM) for immunophenotyping using polychromatic flow cytometry. This methodology can be used to ensure that cell integrity and phenotype stability are not altered through cryopreservation and extended storage. For this analysis, HPBMC were isolated from 7 healthy individuals, and 11‐color flow cytometry was performed on freshly isolated samples as well as samples cryopreserved for short‐ and long‐term periods. There is no significant difference in the percentage of cells expressing the CSM CD3, CD4, CD8, CD45RO, CD16, CD19, or CD56 between freshly isolated and cryopreserved HPBMC. Hence, cryopreservation of HPBMC does not influence the phenotype of distinct cellular subsets in isolated mononuclear cells. This protocol for HPBMC isolation, cryopreservation, and thawing of HPBMC is intended for long‐term studies of large cohorts requiring sample shipment and subsequent batch analysis. © 2017 by John Wiley & Sons, Inc.

Keywords: HPBMC; phenotyping; cell surface markers; immune cell subsets; flow cytometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of HPBMC from Whole Blood
  • Basic Protocol 2: Cryopreservation of HPBMC
  • Basic Protocol 3: Thawing of Cryopreserved HPBMC
  • Basic Protocol 4: Staining of HPBMC for Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of HPBMC from Whole Blood

  Materials
  • Fico/Lite‐LymphoH (e.g., Atlanta Biologicals, cat. no. I40150)
  • Dulbecco's phosphate‐buffered saline (DPBS) without Ca2+ or Mg2+ (e.g., Sigma‐Aldrich, cat. no. D8537)
  • 15‐ and 50‐ml conical centrifuge tubes, sterile
  • Vacuum blood collection tubes, sterile
  • 5‐, 10‐, and 25‐ml serological pipettes, sterile
  • Transfer pipettes, sterile
  • Refrigerated centrifuge
  • Automated or manual cell counter (e.g., Nexcelom Cellometer Auto 2000)
  • Additional reagents and equipment for blood collection (see Bierer & Strober, ) and for determining cell viability (see Johnson, Nguyen, & Coder, )
CAUTION: All waste should be handled as biohazardous material and should be disposed of accordingly. It is recommended to wear appropriate personal protective equipment such as gloves, eye protection, and a disposable lab coat while working with human blood and blood products, including cells.NOTE: Wash, as used in this protocol, refers to the process of centrifuging the sample as directed, aspirating or pouring off the supernatant, leaving the pellet behind, and resuspending the pellet in the next specified solution.ΝΟΤΕ: Many of the steps, such as layering and collection, can be done with transfer, serological, or micropipettes, and it is up to the experimenter to identify which works best and is the most comfortable.

Basic Protocol 2: Cryopreservation of HPBMC

  Materials
  • Freshly isolated HPBMC with viabilities >80% (see protocol 1)
  • Isopropyl alcohol
  • Freezing media (pair A + B; e.g., Athena Enzyme Systems, cat. no. 0406)
  • 2‐ml cryovials (e.g., Nalgene, cat. no. 5000‐0020 or equivalent)
  • Freezing container (e.g., Nalgene Mr. Frosty, Sigma‐Aldrich, cat. no. C1562)
  • Refrigerated centrifuge
  • −80°C freezer
  • Liquid nitrogen vapor phase storage Dewar

Basic Protocol 3: Thawing of Cryopreserved HPBMC

  Materials
  • cRPMI medium (see recipe)
  • Cryopreserved samples (see protocol 2)
  • 15‐ml conical centrifuge tube, sterile
  • 37°C water bath
  • Dry ice
  • Styrofoam container
  • Transfer pipettes, sterile
  • Centrifuge
  • Automated or manual cell counter (e.g., Nexcelom Cellometer Auto 2000)
CAUTION: Cryogenic samples are held in liquid nitrogen at very cold temperatures (less than or equal to −196°C). Cryovials may sometimes explode due to liquid nitrogen leaking into the vial and causing an increase in internal pressure due to temperature change and release of gas. Even when they do not explode, they may be under pressure when the cap is opened. To avoid dispersion of mist as a result of the pressure, cover the vial with a paper towel to loosen the vial lid, releasing any pressure while capturing any mist. Furthermore, it is important to wear safety equipment such as eye protection, lab coat, gloves, and face shield to prevent exposure or abrasions due to exploding vials.ΝΟΤΕ: Wash, as used in this protocol, refers to the process of centrifuging the sample as directed, aspirating or pouring off the supernatant, leaving the pellet behind, and resuspending the pellet in the next specified solution.

Basic Protocol 4: Staining of HPBMC for Flow Cytometry

  Materials
  • Cells to be stained (see protocol 2)
  • Staining buffer (see recipe)
  • Brilliant Stain Buffer (e.g., BD Biosciences, cat. no. 563794)
  • Antibodies of interest
  • FVS
  • Cytofix buffer (e.g., BD Biosciences, cat. no. 554655)
  • Anti‐mouse Ig, κ/Negative Control Compensation Particles Set (e.g., BD Biosciences, cat. no. 552843)
  • Refrigerated centrifuge with plate adapters for 96‐well cluster tubes
  • 96‐well (1.2‐ml) polypropylene cluster tubes in rack (e.g., Corning, cat. no. 4410)
  • Microcentrifuge tubes or other appropriate tubes for mixing antibody cocktail
  • Paper towels (Wypall L40; e.g., Fisher Scientific, cat. no. 19‐042‐427)
  • 12 × 75 mm (5‐ml) polystyrene round‐bottom tubes (e.g., Falcon, cat. no. 352008)
  • Dual position snap caps for 12 × 75 mm tube (e.g., Falcon, cat. no. 352032)
  • Vortex
  • Ice and ice bucket
  • Aluminum foil
  • Flow cytometer (e.g., LSRFortessa) with four laser capability
CAUTION: This protocol involves live human cells and formaldehyde. All waste should be handled as biohazardous material and should be disposed of accordingly. It is recommended to wear appropriate personal protective equipment such as gloves, eye protection, and a disposable lab coat while working with human blood and blood products, including cells.
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Figures

Videos

Literature Cited

Literature Cited
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