Fusion of Myeloma Cells with Immune Spleen Cells

Steven A. Fuller1, Miyoko Takahashi1, John G.R. Hurrell1

1 Allelix Inc., Mississauga, Ontario
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 11.7
DOI:  10.1002/0471142727.mb1107s01
Online Posting Date:  May, 2001
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Abstract

Liquid nitrogen storage is the method of choice for long‐term safekeeping of hybridoma cell lines. Frozen aliquots of originally isolated hybridomas provide insurance against loss of antibody production and vigor during culture. There are many variations of cell freezing methods in use. However, for freezing and recovering hybridomas and lymphoid cells in general, the protocol described in this unit is simple and has been successful.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • Any strain immunized mouse (unit 11.3)
  • Sp2/0 murine myeloma cells in active log phase ( Am. Type Culture Collection #CRL 1581; unit 11.5)
  • Diethyl ether
  • 70% ethanol
  • recipeDulbecco modified Eagle medium (DMEM) with supplements
  • recipeSterile polyethylene glycol (PEG) solution
  • HAT medium (unit 11.6)
  • recipeHT medium
  • Phosphate‐buffered saline (PBS; appendix 22)
  • 15‐ and 50‐ml centrifuge tubes
  • Glass desiccator or metal can with lid
  • Dissecting board
  • 10.5‐cm scissors ( Irex #IR‐105), sterile
  • 10.5‐cm forceps ( Irex #IR‐1393), sterile
  • 60‐ and 100‐mm petri dishes
  • Stainless‐steel strainer (Cellector; GIBCO #1985‐8500), sterile
  • 3‐cc glass syringes with 26‐G needle
  • 5‐ml serological pipets
  • 37° C water bath
  • Stopwatch
  • 8% CO 2‐in‐air gas mixture
  • Humidified CO 2 incubator
  • Polyvinyl or polystyrene 96‐well microtiter plates
  • Inverted microscope
  • Additional reagents and equipment for estimating cell viability by trypan blue exclusion (unit 11.5) and for detection of antibodies (unit 11.4)
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Figures

Videos

Literature Cited

Literature Cited
   Gefter, M.L., Margulies, D.H., and Scharff, M.D. 1977. A simple method for polyethylene glycol‐promoted hybridization of mouse myeloma cells. Somat. Cell Genet. 3:231‐236.
   Kohler, G. and Milstein, C. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495‐497.
   Oi, V.T. and Herzenberg, L.A. 1980. Immunoglobulin‐producing hybrid cell lines. In Selected Methods in Cellular Immunology (B.B. Mishell and S.M. Shiigi, eds.) pp. 351‐372. W.H. Freeman, San Francisco.
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