High-throughput fluorescence-based isolation of live C. elegans larvae.
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.
Department of Biology and Center for Genomics and Systems Biology, New York University, New York, USA. afernandez@fairfield.edu
Anita G
AG
Fernandez
Bastiaan O R
BO
Bargmann
Emily K
EK
Mis
Mark L
ML
Edgley
Kenneth D
KD
Birnbaum
Fabio
F
Piano
D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000235:genetics; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D006720:Homozygote; D007814:Larva
D049452:Green Fluorescent Proteins