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A method for culturing chick melanocytes: The effect of BRL-3A cell conditioning and related additives

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Summary

A method for growing chick embryo melanocytes is described that utilizes medium conditioned by Buffalo Rat liver (BRL-3A) cells. The dissected trunk region of each 72 h (Stages 14 to 19) embryo produces approximately 200,000 melanocytes (purity, 80%) when processed and cultured for 8 d. Thus, a typical experiment involving 20 embryos would produce a total of 4 × 106 melanocytes. Choice of serum, serum concentration, and cell density were determined experimentally. Partially purified multiplication stimulating activity (MSA) from BRL-3A cells and insulin were also tested as medium additives. MSA was not stimulatory, whereas insulin gave a positive response in 2% but not 10 or 0% serum. The final protocol used a modified F12 medium with 10% bovine calf serum conditioned by BRL-3A cells. Cultures were fed every other day. Small colonies of cells became evident by culture Day 3 and increased rapidly to Day 5 when pigmentation became obvious. Colony size continued to increase but more slowly from Days 5 to 8, whereas pigmentation increased rapidly and maximized on Day 8. There is a factor, or factors, present in BRL-3A conditioned medium that stimulates embryonic chick melanocytes to divide preferentially over contaminating cell types. This results in cultures that can provide adequate numbers and purity for biochemical studies.

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Authors and Affiliations

  1. School of Life Sciences, University of Nebraska, 68588, Lincoln, Nebraska

    Barbara Giss, Jane Antoniou, Gary Smith & John Brumbaugh

Authors
  1. Barbara Giss
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  2. Jane Antoniou
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  3. Gary Smith
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  4. John Brumbaugh
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Additional information

This work was supported by National Institutes of Health grants GM18969 to J. B. and CA17620 to G. S.

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Giss, B., Antoniou, J., Smith, G. et al. A method for culturing chick melanocytes: The effect of BRL-3A cell conditioning and related additives. In Vitro Cell.Dev.Biol.-Plant 18, 817–826 (1982). https://doi.org/10.1007/BF02796322

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  • DOI: https://doi.org/10.1007/BF02796322

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