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Automated sorting of live C. elegans using laFACS

Nature Methods volume 7, pages 417–418 (2010)Cite this article

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Although a FACS machine is designed to sort single cells, a few modifications enabled us to sort ∼0.25-mm-long L1 C. elegans. First, we used a reduced drop-drive frequency of ∼16.4 kHz to not damage the larvae. We also used a 100-μm nozzle and set a gate to capture events with a high forward-scatter signal (Fig. 1b), confining our collections to larger objects (Supplementary Methods). After worm sorting, the same FACS machine could be used for several other applications, including sorting of yeast, Drosophila melanogaster cells, mammalian cells and plant protoplasts. The worm sort had no impact on these applications, and the worm-specific FACS machine modifications were easily reversed to accommodate single-cell applications.

We used laFACS to collect mel-28 homozygous worms from a mixed population. mel-28 homozygous hermaphrodites derived from heterozygous mothers appear phenotypically indistinguishable from wild-type worms but produce only inviable progeny. This selection is usually performed manually and thus is not amenable to large-scale applications. We first generated a GFP-marked strain in which the mel-28(t1684) mutation is balanced over a chromosome bearing lag-2::GFP6 (Fig. 1a). We sorted L1 worms, collecting GFP-negative mel-28(t1684) homozygotes. We selected GFP-negative larvae on the basis of the ratio of green (GFP fluorescence; 530/30 nm) to red (red-spectrum autofluorescence; 610/20 nm) signal (Fig. 1c,d). We separated GFP-positive and GFP-negative worms (Supplementary Fig. 1). Typically, from a population of 600,000 larvae, we retrieved ∼130,000 healthy worms after one sort. This first-pass population was ∼95–98% homozygous (n > 1,000, verified by microscopy). A second sort recovered an essentially 100% pure population of about 100,000 homozygous larvae (n > 20,000, verified by microscopy and genetic analysis). This scheme can be easily adapted for the majority of C. elegans genes using available balancers.

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Figure 1: laFACS of GFP-negative L1 larvae.

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Acknowledgements

This work was partly funded by the US National Institute of Child Health and Human Development (R01HD046236) and the National Human Genome Research Institute (U01 HG004276) to F.P., the National Institutes of Health (R01GM078279-01) to K.D.B. and by the National Science Foundation (0827858) to A.G.F. Nematode strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health National Center for Research Resources.

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

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

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

    Anita G Fernandez

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  1. Anita G Fernandez
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  2. Emily K Mis
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Correspondence to Anita G Fernandez.

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Supplementary Figures 1 and 2, Supplementary Table 1, Supplementary Methods (PDF 686 kb)

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Fernandez, A., Mis, E., Bargmann, B. et al. Automated sorting of live C. elegans using laFACS. Nat Methods 7, 417–418 (2010). https://doi.org/10.1038/nmeth.f.304

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