Depletion of Accessory Cells by Adherence to Sephadex G‐10

Karen S. Hathcock1

1 National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.6
DOI:  10.1002/0471142735.im0306s08
Online Posting Date:  May, 2001
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Abstract

Accessory cells such as macrophages and other antigen‐presenting cells (APC) in suspensions of mouse spleen and lymph node are removed by means of their preferential adherence to Sephadex G‐10 polymers. Unlike the use of nylon wool columns to remove B and accessory cells (UNIT), Sephadex G‐10 columns are employed when the main purpose is to yield functional populations of T cells and B cells (by depleting mostly macrophages and other APC). The following technique is simple, fast, and reproducible; however, it results in some retention of B cells on the resin. The basic protocol in this unit involves assembling the column, loading and equilibrating the sterile Sephadex slurry, and passing the cells through the column followed by collection and analysis of eluate. Two support protocols describe preparation of the Sephadex G‐10 slurry for use in the column by fining and sterilization, plus recycling resin from used columns.

     
 
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Table of Contents

  • Support Protocol 1: Preparation of Sterile Sephadex G‐10 Slurry
  • Support Protocol 2: Recycling Used Sephadex G‐10
  • Commentary
     
 
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Materials

Basic Protocol 1:

  Materials
  • Complete RPMI‐5 medium ( appendix 2A)
  • Sephadex G‐10 slurry (one bottle per column; see protocol 2Support Protocol), 37°C and sterile
  • 35‐ml disposable syringe
  • Ring stand
  • 3‐way disposable stopcock
  • 16‐G needle
  • 5 to 15 mg nylon wool ( protocol 2unit 3.2), sterile
  • 50‐ml conical tube
  • Sorvall H‐1000B rotor (or equivalent)
Note: Use sterile technique and maintain cells, medium, and one bottle of sterilized Sephadex G‐10 at 37°C throughout this procedure.

Support Protocol 1: Preparation of Sterile Sephadex G‐10 Slurry

  Additional Materials
  • Sephadex G‐10 resin (Pharmacia)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Suction apparatus (e.g., tubing attached to vacuum flask)
  • 125‐ml autoclavable bottles (e.g., Wheaton)

Support Protocol 2: Recycling Used Sephadex G‐10

  Additional Materials
  • Used Sephadex G‐10 resin (see protocol 1basic protocol)
  • 1% sodium dodecyl sulfate (SDS) in PBS ( appendix 2A)
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Figures

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Literature Cited

Literature Cited
   Ly, I. A. and Mishell, R. I. 1974. Separation of mouse spleen cells by passage through columns of Sephadex G‐10. J. Immunol. Methods. 5:239‐247.
Key Reference
   Hathcock, K. S., Singer, A., and Hodes, R. J. 1981. Passage over Sephadex G‐10 columns.. In Manual of Macrophage Methodology: Collection, Characterization and Function (H. H. Herskowitz, H. T. Holden, J. A. Eelanti and A. Ghaffer, eds.) pp. 127‐133. Marcel Dekker, New York.
  A succinct review of the conditions and applications of this technique.
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