Freezing and Recovery of Hybridoma Cell Lines

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

1 Allelix Inc., Mississauga, Ontario, Canada
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 11.9
DOI:  10.1002/0471142727.mb1109s01
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

  • Basic Protocol 1: Freezing of Cell Lines
  • Basic Protocol 2: Recovery of Frozen Cell Lines
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Freezing of Cell Lines

  Materials
  • recipeFreezing medium
  • 95% ethanol
  • 10% glycerol
  • Cryotubes ( Nunc #3‐63401)
  • 15‐ml plastic centrifuge tubes
  • Liquid N 2 freezer
  • Additional reagents and equipment for estimating cell viability by trypan blue exclusion (unit 11.5)
Ideally, hybridoma cells to be frozen should be in the log phase of growth (as described for the Sp2/0 myeloma cells, unit 11.5, step 5).

Basic Protocol 2: Recovery of Frozen Cell Lines

  Materials
  • Complete culture medium (as described for Sp2/0 cells, unit 11.5)
  • 37°C water bath
  • Alcohol swabs
  • 15‐ml centrifuge tubes
  • 25‐cm2 tissue culture flask (Costar or Falcon)
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Figures

Videos

Literature Cited

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