Preparation of Myeloma 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 11.5
DOI:  10.1002/0471142727.mb1105s18
Online Posting Date:  May, 2001
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Abstract

In this unit, myeloma cells are cultured to ensure their sensitivity to the HAT selection medium used after cell fusion. Cell culture conditions are adjusted such that the Sp2/0 cells are in the log phase of growth and exhibit high viability at the time of collection for fusion. A support protocol is provided to determine the number of viable cells present in the cell culture.

     
 
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Table of Contents

  • Support Protocol 1: Cell Viability Test by Trypan Blue Exclusion
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • Sp2/0 murine myeloma cell line ( ATCC #CRL 1581)
  • recipeComplete culture medium
  • 20 µg/ml 8‐azaguanine
  • Tissue culture flasks, 25 cm2 or 75 cm2
  • 8% CO 2‐in‐air gas mixture
  • Humidified 37°C, 8% CO 2 incubator
  • Inverted microscope

Support Protocol 1: Cell Viability Test by Trypan Blue Exclusion

  Additional Materials
  • Phosphate‐buffered saline (PBS; appendix 22) or serum‐free recipecomplete culture medium
  • 0.4% trypan blue solution
  • Binocular microscope
  • Hemacytometer
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Figures

Videos

Literature Cited

Literature Cited
   Kearney, J.F., Radbruch, A., Liesegang, B., and Rajewsky, K. 1979. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody‐secreting hybrid cell lines. J. Immunol. 123:1548‐1550.
   Kohler, G. and Milstein, C. 1976. Fusion between immunoglobulin‐secreting and nonsecreting myeloma cell lines. Eur. J. Immunol. 6:511‐519.
   Schulman, M., Wilde, C.D., and Kohler, G. 1978. A better cell line for making hybridomas secreting specific antibodies. Nature 276:269‐270.
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