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High-throughput fluorescence-based isolation of live C. elegans larvae

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Abstract

For the nematode Caenorhabditis elegans, automated selection of animals of specific genotypes from a mixed pool has become essential for genetic interaction or chemical screens. To date, such selection has been accomplished using specialized instruments. However, access to such dedicated equipment is not common. Here we describe live animal fluorescence-activated cell sorting (laFACS), a protocol for automatic selection of live first larval stage (L1) animals using a standard FACS system. We show that FACS can be used for the precise identification of GFP-expressing and non-GFP-expressing subpopulations and can accomplish high-speed sorting of live animals. We have routinely collected 100,000 or more homozygotes from a mixed starting population within 2 h, and with greater than 99% purity. The sorted animals continue to develop normally, making this protocol ideally suited for the isolation of terminal mutants for use in genetic interaction or chemical genetic screens.

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Figure 1: Strain PF405 genetics.
Figure 2: Strain PF405 phenotypes.
Figure 3: Preparation of C. elegans for laFACS.
Figure 4: Sorting C. elegans with laFACS.

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Acknowledgements

We thank E. Andersen, D.G. Moerman and R. Waterston for sharing unpublished data; P.-L. Ip, J. Lucas and K. Erikson for technical assistance; and S.D. Weatherbee for critically reviewing the manuscript. Funding sources included the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD; R01HD046236) and the National Human Genome Research Institute (NHGRI; U01 HG004276) to F.P., the US National Institutes of Health (NIH; R01GM078279-01) to K.D.B. and the National Science Foundation (0827858) and Fairfield University start-up funds to A.G.F. Nematode strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR).

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Author notes

  1. Anita G Fernandez and Bastiaan O R Bargmann: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biology and Center for Genomics and Systems Biology, New York University, New York, USA

    Anita G Fernandez, Bastiaan O R Bargmann, Emily K Mis, Kenneth D Birnbaum & Fabio Piano

  2. Department of Biology, Fairfield University, Fairfield, Connecticut, USA

    Anita G Fernandez

  3. Department of Cell and Developmental Biology, University of California San Diego, La Jolla, California, USA

    Bastiaan O R Bargmann

  4. Biotechnology Laboratory and Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada

    Mark L Edgley

Authors
  1. Anita G Fernandez
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  2. Bastiaan O R Bargmann
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  3. Emily K Mis
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  4. Mark L Edgley
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  5. Kenneth D Birnbaum
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  6. Fabio Piano
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Contributions

F.P. and A.G.F. initially developed the idea; B.O.R.B. and A.G.F. developed the FACS conditions; and F.P., A.G.F., B.O.R.B., E.K.M., M.L.E. and K.D.B. contributed to the realization of the protocol.

Corresponding authors

Correspondence to Anita G Fernandez, Bastiaan O R Bargmann or Fabio Piano.

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The authors declare no competing financial interests.

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Fernandez, A., Bargmann, B., Mis, E. et al. High-throughput fluorescence-based isolation of live C. elegans larvae. Nat Protoc 7, 1502–1510 (2012). https://doi.org/10.1038/nprot.2012.084

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