Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)

Ke Lan1, Subhash C. Verma1, Masanao Murakami1, Bharat Bajaj1, Erle S. Robertson1

1 University of Pennsylvania Medical School, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Microbiology
Unit Number:  Appendix 4C
DOI:  10.1002/9780471729259.mca04cs6
Online Posting Date:  August, 2007
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

In this appendix, several basic methods are described for preparation of primary B lymphocyte. Curr. Protoc. Microbiol. 6:A.4C.1‐A.4C.9. © 2007 by John Wiley & Sons, Inc.

Keywords: B lymphocyte; sheep blood cell; T‐cell rosetting; CD19

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Mononuclear Cells by Ficoll‐Hypaque Gradient Centrifugation
  • Basic Protocol 2: Positive Selection of B Lymphocytes from PBMCs Using CD19+ Microbeads
  • Basic Protocol 3: Rapid One‐Step Immunomagnetic Separation of B Lymphocytes from PBMC
  • Basic Protocol 4: Enrichment of B Cells from PBMC by Removal of T Cells Using 2‐Aminoethylisothiouronium Bromide (AET)–Treated Sheep Red Blood Cell Rosetting
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Isolation of Mononuclear Cells by Ficoll‐Hypaque Gradient Centrifugation

  Materials
  • 1000 U/ml heparin
  • Ficoll or Lymphoprep solution (Amersham Biosciences)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • RPMI‐1640 supplemented with 2 mM glutamine and 5% FBS
  • Alcohol swabs
  • 60‐ml syringe
  • Tourniquet
  • 18‐G needle
  • Vacutainer blood collection system
  • 50‐ml centrifuge tube
  • 10‐ml sterile pipet
IMPORTANT NOTE: Blood should be obtained from the patient by an experienced phlebotomist or a licensed physician. IRB approval is necessary to obtain blood from a healthy volunteer with an approved consent form.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique must be used accordingly.NOTE: Figure demonstrates some of the needed materials for this procedure.

Basic Protocol 2: Positive Selection of B Lymphocytes from PBMCs Using CD19+ Microbeads

  Materials
  • Staining buffer (see recipe), ice‐cold
  • Anti‐CD19 antibody: Purified mouse anti–human CD19 antibody (BD Pharmingen)
  • Human Fcγ‐receptor blocking antibody
  • Paramagnetic microbeads conjugated to appropriate secondary antibody
  • 30‐µm nylon mesh
  • Separation tube
  • Powerful magnet
  • Fluorescent‐activated cell sorter (FACS)
  • Additional reagents and equipment for isolating lymphocytes from whole blood (see protocol 1) and counting cells using a hemacytometer ( appendix 4A).

Basic Protocol 3: Rapid One‐Step Immunomagnetic Separation of B Lymphocytes from PBMC

  Materials
  • Staining buffer (see recipe)
  • 30‐µm nylon mesh
  • Microbeads mix (Miltenyi Biotech GmbH)
  • Magnetic separator (Miltenyi Biotech GmbH)
  • Magnetic separation column (Miltenyi Biotech GmbH)
  • Plunger (Miltenyi Biotech GmbH)
  • Fluorescence‐activated cell sorter (FACS)
  • Additional reagents and equipment for isolating lymphocytes from whole blood (see protocol 1)
NOTE: Figure shows the assembled MACS positive‐selection apparatus.

Basic Protocol 4: Enrichment of B Cells from PBMC by Removal of T Cells Using 2‐Aminoethylisothiouronium Bromide (AET)–Treated Sheep Red Blood Cell Rosetting

  Materials
  • Sheep red blood cells (SRBC; usually purchased from Biowhittaker)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Peripheral blood mononuclear cells (PBMC) in complete RPMI‐10 medium (see protocol 1)
  • RPMI‐1640 supplemented with 2 mM glutamine and 5% FBS
  • 0.14 M 2‐aminoethylisothirouroniumuronium hydrobromide solution (AET; see recipe)
  • Tris‐buffered NH 4Cl, pH 7.5
  • Fetal bovine serum (FBS; appendix 2A)
  • Centrifuge with a swinging‐bucket rotor
  • 37°C water bath
  • 5‐ml pipet, sterile
  • Additional reagents and solutions for counting cells ( appendix 4A)
NOTE: Prepare AET treated SRBC in a sterile manner.NOTE: To prepare a large amount of AET treated SRBC, increase the number of tubes in step one.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure a0.4C.1 Components including syringe, vacutainer, tourniquet, heparin, alcohol swab, band aid, and gauze for bloodletting.
  •   FigureFigure a0.4C.2 (A) Scheme showing blood separation by Ficoll gradient. (B) An actual separation of blood after Ficoll gradient centrifuge.
  •   FigureFigure a0.4C.3 Schematic showing various steps in positive selection of CD19+ B lymphocytes.
  •   FigureFigure a0.4C.4 Picture showing the assembled Miltenyi MACS positive‐selection apparatus. The column is vertically mounted in a strong magnet.

Videos

Literature Cited

Literature Cited
   Fong, S., Tsoukas, C.D., Pasquali, J.L., Fox, R.I., Rose, J.E., Raiklen, D., Carson, D.A., and Vaughan, J.H. 1981. Fractionation of human lymphocyte subpopulations on immunoglobulin coated Petri dishes. J. Immunol. Methods 44:171‐182.
   Madsen, M. and Johnsen, H.E. 1979. A methodological study of E‐rosette formation using AET‐treated sheep red blood cells. J. Immunol. Methods 10:61‐74.
   Strelkauskas, A.J., Teodorescu, M., and Dray, S. 1975. Enumeration and isolation of human T and B lymphocytes by rosette formation with antibody‐coated erythrocytes. Clin. Exp. Immunol. 22:62‐71.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library