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Fractionation of T and B Cells Using Magnetic Beads

Angela M. Thornton1

1National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland

Publication Name: 
Current Protocols in Immunology
Unit Number: 
Unit 3.5A
DOI: 
10.1002/0471142735.im0305as57
Online Posting Date: 
November, 2003
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Angela Thornton

Abstract

This unit describes the purification of mouse T cells, B cells, and T cell subsets using magnetic bead separation. Isolation of cell subsets using magnetic beads is quick, simple, and reliable and can result in high yields of very pure cells.

Keywords: mouse; T cells; B cells; suppressor T cells; purification/isolation

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Automated Isolation of Total T Cells, CD4+ T Cells, or CD8+ T Cells Using the Automacs System
  • Alternate Protocol 1: Manual Isolation of Total T Cells, CD4+ T Cells, or CD8+ T Cells Using Magnetic Columns
  • Basic Protocol 2: Automated Isolation of B Cells Using the Automacs System
  • Alternate Protocol 2: Manual Isolation of B Cells Using Magnetic Columns
  • Basic Protocol 3: Automated Isolation of Accessory Cells Using the Automacs System
  • Alternate Protocol 3: Manual Isolation of Accessory Cells Using Magnetic Columns
  • Basic Protocol 4: Automated Isolation of CD4+CD25+Suppressor Cells Using the Automacs System
  • Alternate Protocol 4: Manual Isolation of CD4+CD25+Suppressor Cells Using Magnetic Columns
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Automated Isolation of Total T Cells, CD4+ T Cells, or CD8+ T Cells Using the Automacs System

 Materials
  • Mice
  • HBSS wash buffer (see recipe)
  • MACS buffer (see recipe)
  • Magnetic beads (Table 3.5A.1; Miltenyi)
  • Complete RPMI-10 (appendix 2A)
  • autoMACS system and columns (Miltenyi)
  • 30-µm nylon mesh (BD) or to a 40-µm preseparation filter (Miltenyi)
  • Additional reagents and solutions for preparation of cell suspensions from mouse lymph nodes or spleens and removal of red blood cells (unit 3.1), and counting cells using a hemocytometer (appendix 3A)
     
    Table 3.5A.1 Magnetic Bead Choices for Mouse T Cell Populations
    Target populationMicrobeads
    Total T cellsCD90 (Thy1.2)
    CD4+ T cellsCD4 (L3T4)
    CD8+ T cellsCD8a (Ly-2)

Alternate Protocol 1: Manual Isolation of Total T Cells, CD4+ T Cells, or CD8+ T Cells Using Magnetic Columns

 Additional Materials (also see Basic Protocol 1)
  • Degassed MACS buffer, 4°C (see recipe)
  • MidiMACS Separation unit (Miltenyi)
  • MACS Multistand (Miltenyi)
  • LS column (Miltenyi)
  • 15-ml tubes, sterile

NOTE: The MidiMACS Separation unit, MACS Multistand, and LS columns can be purchased collectively in the MidiMACS Starting Kit (Miltenyi).

Basic Protocol 2: Automated Isolation of B Cells Using the Automacs System

 Materials
  • Mice
  • HBSS wash buffer (see recipe)
  • MACS buffer (see recipe)
  • CD45R (B220) magnetic beads (Miltenyi)
  • Complete RPMI-10 (appendix 2A)
  • autoMACS system and columns (Miltenyi)
  • 30-µm nylon mesh (BD) or a 40-µm preseparation filter (Miltenyi)
  • Additional reagents and solutions for preparation of cell suspensions from mouse spleens and removal of red blood cells (unit 3.1), and counting cells using a hemocytometer (appendix 3A)

Alternate Protocol 2: Manual Isolation of B Cells Using Magnetic Columns

 Additional Materials (also see Basic Protocol 2)
  • Degassed MACS buffer, 4°C (see recipe)
  • MidiMACS Separation unit (Miltenyi)
  • MACS Multistand (Miltenyi)
  • LS columns (Miltenyi)
  • 15-ml tubes, sterile

NOTE: The MidiMACS Separation unit, MACS Multistand, and LS columns can be purchased collectively in the MidiMACS Starting Kit (Miltenyi).

Basic Protocol 3: Automated Isolation of Accessory Cells Using the Automacs System

 Materials
  • Mice
  • HBSS wash buffer (see recipe)
  • MACS buffer (see recipe)
  • CD90 (Thy 1.2) magnetic beads (Miltenyi)
  • Complete RPMI-10 (appendix 2A)
  • autoMACS system and columns (Miltenyi)
  • 30-µm nylon mesh filter (BD) or a 40-µm preseparation
  • Additional reagents and solutions for preparation of cell suspensions from mouse spleens and removal of red blood cells (unit 3.1), and counting cells using a hemocytometer (appendix 3A)

Alternate Protocol 3: Manual Isolation of Accessory Cells Using Magnetic Columns

 Additional Materials (also see Basic Protocol 3)
  • Degassed MACS buffer, 4°C (see recipe)
  • MidiMACS Separation unit (Miltenyi)
  • MACS Multistand (Miltenyi)
  • LD columns (Miltenyi)
  • 15-ml tube, sterile

NOTE: The MidiMACS Separation unit, MACS Multistand, and LD columns can be purchased collectively in the LD MidiMACS Starting Kit (Miltenyi).

Basic Protocol 4: Automated Isolation of CD4+CD25+Suppressor Cells Using the Automacs System

 Materials
  • Mice
  • HBSS wash buffer (see recipe)
  • MACS buffer (see recipe)
  • CD8a (Ly-2) magnetic beads (Miltenyi)
  • Binding buffer (see recipe)
  • Biotin-anti-CD25 (7D4) (PharMingen)
  • Streptavidin-PE (PharMingen)
  • Anti-PE microbeads (Miltenyi)
  • Complete RPMI (appendix 2A)
  • Small mouse CD3+ T cell enrichment columns and 1× buffer (R & D Systems)
  • 15-ml tubes, sterile
  • 30-µm nylon mesh filter (BD) or a 40-µm preseparation filter (Miltenyi)
  • autoMACS system and columns (Miltenyi)
  • Additional reagents and solutions for preparation of cell suspensions from mouse spleens and removal of red blood cells (unit 3.1), and counting cells using a hemocytometer (appendix 3A)

Alternate Protocol 4: Manual Isolation of CD4+CD25+Suppressor Cells Using Magnetic Columns

 Additional Materials (also see Basic Protocol 4)
  • Degassed MACS buffer, 4°C (see recipe)
  • MidiMACS Separation Unit (Miltenyi)
  • MACS Multistand (Miltenyi)
  • LD columns (Miltenyi)
  • 15-ml tube, sterile
  • LS columns (Miltenyi)

NOTE: The MidiMACS Separation unit, MACS Multistand, and LS columns can be purchased collectively in the MidiMACS Starting Kit, while the equivalent with LD columns can be purchase collectively in the LD MidiMACS Starting Kit (Miltenyi).

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