Automated sorting of live c. Elegans using lafacs

Access through your institution Buy or subscribe 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::GFP_6 (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. This is a preview

of subscription content, access via your institution ACCESS OPTIONS Access through your institution Subscribe to this journal Receive 12 print issues and online access $259.00 per year only

$21.58 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout

ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support REFERENCES * Stoeckius, M. et al. _Nat. Methods_ 6, 745–751 (2009).

Article  CAS  Google Scholar  * Fernandez, A.G. & Piano, F. _Curr. Biol._ 16, 1757–1763 (2006). Article  CAS  Google Scholar  * Galy, V., Askjaer, P., Franz, C., Lopez-Iglesias, C. &

Mattaj, I.W. _Curr. Biol._ 16, 1748–1756 (2006). Article  CAS  Google Scholar  * Rasala, B.A., Orjalo, A.V., Shen, Z., Briggs, S. & Forbes, D.J. _Proc. Natl. Acad. Sci. USA_ 103,

17801–17806 (2006). Article  CAS  Google Scholar  * Franz, C. et al. _EMBO Rep._ 8, 165–172 (2007). Article  CAS  Google Scholar  * Edgley, M.K., Baillie, D.L., Riddle, D.L. & Rose, A.M.

in _WormBook_ (The _C. elegans_ research community, 2006). Google Scholar  * Kamath, R.S. et al. _Nature_ 421, 231–237 (2003). Article  CAS  Google Scholar  Download references

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. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * 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 * Department of

Biology, Fairfield University, Fairfield, Connecticut, USA Anita G Fernandez Authors * Anita G Fernandez View author publications You can also search for this author inPubMed Google Scholar

* Emily K Mis View author publications You can also search for this author inPubMed Google Scholar * Bastiaan O R Bargmann View author publications You can also search for this author

inPubMed Google Scholar * Kenneth D Birnbaum View author publications You can also search for this author inPubMed Google Scholar * Fabio Piano View author publications You can also search

for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Anita G Fernandez. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial

interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Figures 1 and 2, Supplementary Table 1, Supplementary Methods (PDF 686 kb) RIGHTS AND PERMISSIONS Reprints

and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Published: 02 May 2010 * Issue Date: June 2010 * DOI: https://doi.org/10.1038/nmeth.f.304 SHARE THIS ARTICLE Anyone you share the

following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer

Nature SharedIt content-sharing initiative